Ceramide accumulation mediates inflammation, cell death and infection susceptibility in cystic fibrosis

Size: px
Start display at page:

Download "Ceramide accumulation mediates inflammation, cell death and infection susceptibility in cystic fibrosis"

Transcription

1 - 1 - Supplementary Information Ceramide accumulation mediates inflammation, cell death and infection susceptibility in cystic fibrosis Volker Teichgräber 1, Martina Ulrich 2, Nicole Endlich 3, Joachim Riethmüller 4, Barbara Wilker 1, Cheyla Conceição De Oliveira Munding 2, Anna M van Heeckeren 5, Mark L Barr 6, Gabriele von Kürthy 7, Kurt W Schmid 8, Michael Weller 7, Burkhard Tümmler 9, Florian Lang 10, Heike Grassme 1, Gerd Döring 2 & Erich Gulbins 1 1 Department of Molecular Biology, Hufelandstrasse 55, University of Duisburg-Essen, Essen, Germany. 2 Institute of Medical Microbiology and Hygiene, Wilhelmstrasse 31, Tübingen, Germany. 3 Department of Anatomy, Friedrich-Loeffler-Str. 23c, University of Greifswald, Greifswald, Germany. 4 Children`s Clinic, Auf dem Schnarrenberg, University of Tübingen, Tübingen, Germany. 5 Case Western Reserve University, Biomedical Research Building 827, Euclid Avenue, Cleveland, Ohio , USA. 6 Department of Cardiothoracic Surgery, University of Southern California, 1520 San Pablo Street, Suite 4300, Los Angeles, California 90033, USA. 7 Department of Neurology, Auf dem Schnarrenberg, University of Tübingen, Tübingen, Germany. 8 Department of Pathology and Neuropathology, Hufelandstrasse 55, University of Duisburg-Essen, Essen, Germany. 9 Medical University School, Carl-Neuberg-Strasse, Hannover, Germany. 10 Department of Physiology, Gmelinstrasse 5, University of Tübingen, Tübingen, Germany. Address correspondence to: Dr. Erich Gulbins, Dept. of Molecular Biology, University of Duisburg Essen, Hufelandstrasse 55, Essen, Tel.: , Fax: , e mail: erich.gulbins@uni due.de

2 - 2 - Supplementary note 1 Sphingosine 1 phosphate and sphingosine levels in CF mice Sphingosine 1 phosphate levels were 2.6 ± 1.1 pmol/mg protein in Cftr MHH compared to 2.8 ± 1.1 pmol/mg protein in wild type mice. Sphingosine concentrations were 4.08 ± 0.25 pmol/mg protein in 24 weeks old Cftr MHH compared to 1.0 ± 0.11 pmol/mg protein in wild type mice (n = 5 each). We also tested whether the uptake of [ 3 H]sphingosine and [ 3 H]sphingosine 1 phosphate is affected by Cftr deficiency in tracheal epithelial cells. More than 95% of the added [ 3 H]sphingosine was converted to [ 3 H]sphingosine 1 phosphate within 5 min after addition to tracheal cells from wild type, Cftr KO and Cftr MHH mice. The uptake of [ 3 H]sphingosine 1 phosphate was reduced by approximately 50% in Cftr deficient epithelial cells (not shown).

3 - 3 - Supplementary Figure 1 Peptamen diet reduces accumulation of ceramide in CF mice We tested whether Peptamen R alters ceramide levels in the lungs of CF mice and fed our Cftr deficient mice, which express a residual activity of Cftr (Cftr MHH strain) or express Cftr in the intestinum as a transgen (Cftr KO strain), with Peptamen R. The data (Supplementary Figure 1a) demonstrated that Peptamen R reduced ceramide levels in lungs of Cftr MHH mice by 92% compared to untreated Cftr MHH mice and in lungs of Cftr KO mice by 86%. Compared to untreated wild type mice, Peptamen R treated Cftr MHH mice indeed revealed reduced ceramide levels. To address the mechanisms that mediate the reduction of ceramide upon Peptamen R feeding, we determined the concentration of cholesterol in the lungs of animals fed with Peptamen R. Cholesterol has been previously shown to critically influence the activity of the Asm and high levels of cholesterol significantly reduce the activity of the Asm 1. Our data demonstrated a 3 fold increase of cholesterol in the lung upon Peptamen R feeding (Supplementary Figure 1b). Simultaneous treatment of CF mice with simvastatin (40 mg/kg/day i.p.), a known blocker of cholesterol biosynthesis 2 prevented the cholesterol accumulation (Supplementary Figure 1b) as well as the reduction of ceramide in the lungs of these animals (Supplementary Figure 1a). Consistent with its effect on ceramide, Peptamen R diet resulted in 60% inhibition of the activity of the Asm in the lung, an effect that was abrogated by treatment with simvastatin (Supplementary Figure 1c). These in vivo data are supported by in vitro studies that show a dose dependent inhibition of the Asm in isolated respiratory epithelial cells incubated with increasing doses of cholesterol (Supplementary Figure 1d).

4 - 4 - Supplementary Fig. 1. High cholesterol levels inhibit acid sphingomyelinase (Asm) activity and normalize ceramide levels in CF mice. (a,b) Feeding of Cftr MHH or Cftr KO mice for 3 weeks with Peptamen R ad libitum reduces ceramide (a) and increases cholesterol (b) concentrations in the lung, effects that are blocked by simvastatin (Sim) treatment. Data represent means ± SD from each 5 independent experiments. (a) *P < 0.05, **P < 0.01, ***P < 0.001, ANOVA; (b) *P < 0.04; Mann Whitney test. (c) Feeding of mice with Peptamen R for 14 d reduced Asm activity in mouse lungs, which was prevented by treatment with simvastatin. (d) Similarly, Asm activity was reduced in isolated epithelial cells, incubated with increasing doses of cholesterol. Displayed are means ± SD of 5 independent experiments each. Significant differences to untreated controls are indicated by asterisks (**P < 0.01, ***P < 0.002; exact Mann Whitney test). References: 1. Bhuvaneswaran, C., Venkatesan, S. & Mitropoulos K.A. Lysosomal accumulation of

5 - 5 - cholesterol and sphingomyelin: evidence for inhibition of acid sphingomyelinase. Eur. J. Cell Biol. 73, (1985). 2. Mol, M.J., Erkelens, D.W., Leuven J.A., Schouten J.A. & Stalenhoef, A.F. Effects of synvinolin (MK 733) on plasma lipids in familial hypercholesterolaemia. Lancet 25, (1986).

6 - 6 - Supplementary note 2 Methods Mouse strains. We bred and housed Cftr tm1unc -Tg (FABPCFTR) (abbreviated Cftr KO ) Jaw mice (Jackson Laboratories) in the vivarium of the University of Duisburg-Essen, Germany. These mice, which are on a mixed background consisting of C57BL/6, FVB/N and 129, are genetically deficient for the mouse equivalent to human CFTR (Cftr), but express human CFTR in the gut under control of a fatty acid binding protein (FABP) promoter, which prevents acute intestinal obstruction 1,2. C3He mice were used as controls. Additionally, B6.129P2(CF/3)-Cftr TgH(neoim)Hgu (abbreviated Cftr MHH ) congenic mice were used 3. The inbred CF strain CF/3-Cftr TgH(neoim)Hgu was established by brother-sister mating from the original Cftr TgH(neoim)Hgu mutant mouse that produces low levels of Cftr 4-6. Then, the congenic Cftr MHH strain was generated by backcrossing the targeted mutation into the B6 inbred background by speed congenic production. Syngenic B6 mice were used as controls. Cftr MHH and control mice were housed both in the Central Laboratory Animal Facility of the Medizinische Hochschule Hannover and the University of Duisburg-Essen, Germany. Furthermore, 12 week old C57BL/6 congenic B6.129P2-Cftr tm1unc mice bearing the S489X mutation in Cftr (Cftr S489X ) were used; Cftr +/+ mice were used as wild type littermate controls. Cftr S489X mice were housed and maintained in the vivarium at Case Western Reserve University, Cleveland, USA 7. Finally, Cftr KO mice were bred with Smpd1 -/- mice that are on a C57BL/6 background. To obtain Cftr KO /Smpd1 +/- mice, the heterozygous offspring was backcrossed to Cftr KO mice for at least 5 generations. The different laboratories, housing the mice in isolator cages, provided a pathogen-free environment. The mice were routinely investigated for microbial infections by bacterial culturing and serology and remained negative during the study period. The hygienic status was repeatedly tested by a panel of common mouse pathogens according to the FELASA recommendations of

7 - 7 - Bacterial lung infection. Mice were challenged with the P. aeruginosa strain 762, obtained from a patient with urosepsis, strain 769, from a patient with pneumonia or the laboratory strain ATCC 14115, obtained from ATCC. Bacteria were grown for 14 h to 14.5 h on fresh (maximally 2 weeks old) Trypticase Soy Agar (TSA) plates (Becton Dickinson, # ). Bacteria were transferred into 40 ml 37 o C pre-warmed Trypticase Soy Broth (TSB) (Becton Dickinson, # ) in Erlenmeyer flasks at an optical density (OD) (550 nm) of 0.225/ml (equals to 5.5 x 10 8 colony forming units (CFU)/ml) and the bacteria were incubated for 60 min at 37 o C with shaking at 125 rpm. The quality and source of the agar plates and the TSB, the shaking speed and the incubation time are critical to obtain reproducible results. Bacteria were centrifuged at 2,800 rpm (1600 x g) and the cell pellet re-suspended in 10 ml prewarmed RPMI-1640, supplemented with 10 mm HEPES, ph 7.3. The bacterial suspension was washed twice and finally re-suspended at 10 8 CFU/20 µl in pre-warmed HEPES/Saline (H/S) consisting of 132 mm NaCl, 20 mm HEPES, ph 7.4, 5 mm KCl, 1 mm CaCl 2, 0.7 mm MgCl 2, and 0.8 mm MgSO 4. Mice were then infected within the next 10 min. We employed a 30-gauge needle of a 1 ml insulin syringe covered with a tightly fitting, smooth plastic tube to avoid injuries in the nose. The mice were anesthesized for sec in ether (in a covered two liter beaker filled with a few paper towels to get a saturated ether athmosphere), the needle was immediately inserted for 2 mm into the nose and the bacteria were carefully injected. Between the 1st and the last mouse infected, we allowed a time interval of maximally 10 min, to exclude a change in viable counts of the bacteria. Usually, we infected not more than 10 mice/experiment. Of note, we never infected more than one mouse per group and the numbers given in the text indicate the number of independent experiments with one mouse each. P. aeruginosa cell numbers were determined in mouse lungs 15, 30, 60 or 120 min after challenge. Lungs were removed, homogenised and lysed for 10 min in 5 mg/ml saponin to release intracellular bacteria. Dilutions of the homogenates were cultured on TSB plates in

8 - 8 - duplicates. Bacterial counts, determined after 18 h, were normalized for lung weight. Additionally, the survival of infected mice was determined for 7 d after challenge. Control experiments reveal that 10 mg/l amitriptyline did not affect growth of the bacteria. Streptococcus pneumoniae strain R6 was grown on sheep blood tryptic soy agar for 12 h, removed from the plate and grown in TSB for 1 h. The mice were intranasally infected with 10 7 CFU and the number of bacteria in the lungs was determined 3 h after infection as above. Drug administration to mouse lungs and pulmonary lavage. We administered 1 ml of recombinant human DNase (rhdnase) (Roche) (25 µg/ml) to the mice using a Pariboy SX nebulizer apparatus and a LL-Nebulizer mask (Pari). The mask was clipped at the sides to cover only the nose and manually pressed on the mouse nose for 20 min. Similarly, we let mice inhale 1 ml of a solution of bafilomycin (1 µm), nigericin (2.5 µm), NH 4 Cl (25 mm), chloroquine (10 µm) and n-oleoylethanolamine (100 µm) in 0.9% NaCl. Optimal drug concentrations were determined in pilot experiments to achieve maximum DNA digestion in the bronchi, or sufficient alkalinisation of vesicles in lung cells and to cause minimal adverse effects such as increased breathing frequency. To obtain mouse lung macrophages, mice were killed and the trachea canuled with a plastic catheter (at least 5 mm inner diameter) connected to a syringe using surgical thread. The thorax was carefully opened, lungs flushed six times with 1 ml of a 0.9% NaCl solution each and the lavage fluid carefully collected by slow suction. Uptake of sphingosine/sphingosine-1-phosphate. We removed the trachea from wild type-, Cftr KO and Cftr MHH mice and opened it after suspending it in RPMI 1640, supplemented with 10 mm HEPES and 10% fetal calf serum (FCS). Then we incubated the trachea with 5 µci [ 3 H]sphingosine (20 Ci/mmol, American Radiolabelled Chemicals) and 2 µm sphingosine or 5 µci [ 14 C]sphingosine-1-phosphate (25 mci/mmol, American Radiolabelled Chemicals) and

9 - 9-2 µm sphingosine-1-phosphate for 45 min at 37 o C as described 9. We washed the trachea in PBS and determined the uptake of the radioactivity by liquid scintillation counting. Asm activity. We homogenized mouse lungs in 250 mm sodium acetate buffer, ph 5.0, supplemented with 1.3 mm EDTA and 1% NP40 employing a dounce homogenisator followed by 3 rounds of sonication (Ultrasonic Processor tip sonicator). An aliquot of the samples was diluted to 0.1% NP40 and incubated with 0.05 µci per sample [ 14 C]sphingomyelin (52 mci/mmol; MP Biomedicals). Prior to addition we dried the substrate, then re-suspended it in 250 mm sodium acetate buffer (ph 4.5, 5.0 or 5.9), supplemented with 1.3 mm EDTA, and 0.1% NP40 and bath sonicated the samples for 10 min. The samples were incubated for 30 min at 37 o C. The enzymatic reaction was terminated by extraction in 4 volumes of CHCl 3 :CH 3 OH (2:1, v/v), the samples were centrifuged and an aliquot of the upper aqueous phase was scintillation counted to determine the release of [ 14 C]phosphorylcholine from [ 14 C]sphingomyelin. Acid ceramidase activity. We lysed mouse lung cells in 200 mm citrate/phosphate buffer (ph 4.5, 5.0 or 5.9), supplemented with 300 mm NaCl, and 1% Triton X-100, on ice for 15 min. After dilution to 0.1% Triton X-100 we added the substrate [ 14 ]C 16 -ceramide (0.1 µci/sample, 55 mci/mmol, ARC). Prior to addition, the substrate was dried, re-suspended in 150 mm NaCl, supplemented with 0.05% Triton X-100, and sonicated to get micelles. The enzyme reaction was performed for 30 min at 37 o C. We terminated the reaction by addition of 20 volumes ethanol, centrifuged the samples, dried the supernatants, re-suspended in CHCl 3 :CH 3 OH (1:1, v/v) and separated lipids on TLC employing CHCl 3 :CH 3 OH:28% ammonium hydroxide (90:20:0.5, v/v/v). The TLC plates were exposed to X-ray films, the substrate and the product were identified by co-migration with standards, removed from the plate and liquid scintillation counted. To determine the reverse Ac activity, the enzyme

10 reaction was performed with 200 µm [ 14 C] lauric acid (50 mci/mmol) and sphingosine (50 nmol/reaction) at ph 4.5, 5.0 and 5.9. Ceramide was identified by TLC and quantified as above. Ceramide measurements. Mouse lungs were removed, transferred into 1 ml CHCl 3 :CH 3 OH:1N HCl (100:100:1, v/v/v) and homogenized 30-times in a Dounce homogenisator and three times for 20 sec each with a tip sonicator. After addition of 200 µl H 2 O, the samples were centrifuged for 5 min at 14,000 rpm. Serum samples were directly added to 1 ml CHCl 3 :CH 3 OH:1N HCl (100:100:1, v/v/v). The lower phase was collected, dried and subjected to alkaline hydrolysis of diacylglycerol in 0.1 N methanolic KOH at 37 o C for 60 min. The samples were re-extracted, the lower phase dried and re-suspended in 20 µl of a detergent solution (7.5% (w/v) n-octylglucopyranoside, 5 mm cardiolipin in 1 mm diethylene-triaminepentaacetic acid). After sonication (10 min), the samples were added to 70 µl of a reaction mixture, containing 10 µl diacylglycerol kinase (GE Healthcare Europe), 0.1 M imidazole/hcl, ph 6.6, 0.2 mm diethylenetriamine-pentaacetic acid, ph 6.6, 70 mm NaCl, 17 mm MgCl 2, 1.4 mm EGTA, 2 mm DTT, 1 µm ATP and 10 µci [ 32 P]γATP. The kinase reaction was performed for 30 min at room temperature and terminated by addition of 1 ml CHCl 3 :CH 3 OH:1N HCl (100:100:1, v/v/v), 170 µl buffered saline solution (135 mm NaCl, 1.5 mm CaCl 2, 0.5 mm MgCl 2, 5.6 mm glucose, 10 mm HEPES), ph 7.2, and 30 µl of a 100 mm EDTA solution. The samples were vortexed and the phases separated. The lower phase was collected, dried, dissolved in 20 µl CHCl 3 :CH 3 OH (1:1, v/v) and separated on Silica G60 TLC plates employing CHCl 3 :CH 3 OH:CH 3 COOH (65:15:5, v/v/v). The TLC-plates were exposed to x-ray films and ceramide spots were identified by co-migration with a C 16 - ceramide standard. Incorporation of [ 32 P] into ceramide was quantified by liquid scintillation counting after removal of the spots from the plates. Ceramide amounts were determined by

11 comparison with a standard curve using C 16 -ceramide as substrate. We performed six independent experiments with each one mouse per group. Cholesterol measurements. Cholesterol was determined using the ECCH-100 kit (BioAssay Systems), following the protocol of the vendor. Briefly, cholesterol esters are converted to cholesterol, cholesterol is then metabolized to cholest-4-ene-3-one and NAD reduced to NADH by cholesterol dehydrogenase. Absorbance at 340 nm was determined to measure cholesterol concentrations. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) of lung sections. We fixed lungs in 4% buffered paraformaldehyde (PFA), ph 7.3, for 36 h and embedded them in paraffin. We de-paraffinized, re-hydrated and treated the samples with 0.1 M citrate buffer, ph 6.0, at 350 W for 5 min in a microwave, washed them in PBS, blocked them in 0.1 M Tris/HCl buffer, ph 7.5, supplemented with 3% bovine serum albumin and 20% FCS and incubated them with fluorescein-coupled dutp and TUNEL enzyme (Roche Diagnostics) in the presence of terminal deoxynucleotidyl transferase. Then, we washed the samples again with PBS, incubated them for 10 min at 70 o C to remove unspecific binding and labeled them with an antibody to FITC, coupled to alkaline phosphatase, in PBS. The signal was then visualized by a colorimetric reaction, the sections were mounted in moviol and analyzed by light microscopy. Ceramide staining. To visualize ceramide in epithelial cells, we isolated large bronchi from the indicated mice, fixed them in 2% PFA, ph 7.3, in PBS for 30 min and scratched ciliated epithelial cells from the tissue. The cells were blocked in PBS, supplemented with 5% FCS, and stained with monoclonal antibodies to ceramide (1:100 dilution, clone 15B4 from Alexis or clone MAS 0010 from Glycobiotech), followed by incubation with a secondary Cy3-

12 labelled antibody against mouse IgM. We analysed the cells on a Leica fluorescence microscope DMIRE2 with an exposure time of 150 ms. All microscopy studies were done at 1000x magnification if not otherwise noted. We collected human epithelial cells by nasal brushings using cotton tips and washed the cells carefully five times in H/S buffer, fixed them in 4% PFA for 15 min and stained them as described above. The fluorescence signal was quantified in areas of 0.5 x 0.5 cm of the fluorescence pictures using the QFluoro software (Leica). We analysed nasal epithelial cells on a DMIRE 2 fluorescence microscope (Leica) with an exposure time of 140 ms. We stained ceramide in mouse lung sections after fixation, embedding in paraffin, dewaxing and re-hydration by incubating the slides in PBS, supplemented with 0.01% Tween 20 for 15 min. The slides were washed in PBS and the tissue was incubated with monoclonal antibodies to ceramide (1:100 dilution, clone MAS 0010) for 45 min at room temperature, followed by incubation with a Cy3-labelled antibody against mouse IgM (1:1000) for 45 min at room temperature. The sections were mounted in moviol and analyzed by fluorescence microscopy as above or by confocal microscopy. Confocal microscopy studies were done with the same microscopy settings on paraffin sections of 16 week old wild type and Cftr MHH mice. FACS studies were performed on crude vesicle preparations from lungs of the indicated mice. Vesicles were stained with Cy3 coupled ceramide antibodies. Analysis was performed on a BD Calibur with standard settings. We stained ceramide in human lung sections using 5 µm cryostat thin sections, prepared from lung tissues of CF and normal individuals. We fixed the sections on glass slides with acetone, washed them with PBS-Tween (0.1%), and pre-incubated them with normal goat serum, 1:10 diluted in PBS-Tween (0.1%). The sections were incubated with monoclonal antibodies to ceramide (Axxora), 1:20 diluted in PBS, at room temperature for 2 h. Then, we washed the sections with PBS-Tween (0.1%) and incubated them with Cy2-labeled goat

13 antibodies to mouse IgM for 1 h at room temperature, followed by three wash steps and an incubation with 2 µg/ml of 4,6-diamidino-2-phenylindole-dihydrochloride (DAPI; Roche Mannheim) in PBS-Tween (0.1%) for 5 min at room temperature. We washed the sections again and embedded them with fluorescence mounting medium. Lung tissues were analysed with a Zeiss Axioplan microscope at an exposure time of 800 ms. We analysed a similar number of airways each in healthy and CF tissues. For ceramide staining by transmission electron microscopy, we studied 16 week old wild type and Cftr MHH mice. We fixed mouse lung tissue (1 mm 3 cubes) with 4% PFA, dehydrated it in a graded ethanol series and embedded it in LR-White (Plano). We performed immunogold double staining on ultrathin sections of 70 nm. After blocking for 1 h, we incubated the sections with monoclonal antibodies to ceramide and cathepsin D specific antibodies at 4 C overnight, followed by four wash steps with PBS-Tween and an incubation with secondary antibodies, i.e. anti-mouse IgM conjugated with 25 nm gold and anti-goat IgG conjugated with 10 nm gold, each diluted 1:25 for 1 h. We washed the samples four times with TBS-Tween and finally with H 2 O, and post-stained them with uranyl acetate. We investigated the sections with an EM 910 microscope (Zeiss). Image documentations were done with Kodak 4489 macro EM film. Corrections of background illumination and contrast enhancement were done in ImageJ (Wayne Rusband, NIH). DNA staining of lung sections and adhesion of P. aeruginosa to DNA. Re-hydrated sections from mouse lungs were incubated for 5 min in 4 µg/ml ethidium bromide, washed in PBS, mounted in moviol and analyzed by fluorescence microscopy. To determine P. aeruginosa adhesion to and growth on DNA, we pre-incubated A549 respiratory cells with 10 µg denatured salmon sperm DNA or medium for 30 min at 37 C. Then we incubated cells with a suspension of 1x 10 7 CFU/ml of P. aeruginosa for 2 h at 37 C. We removed the

14 supernatant, fixed the cells with 4% formaldehyde, and stained P. aeruginosa with a Cy3- coupled antibody to P. aeruginosa. We visualized eukaryotic cells with DAPI. Determination of neutrophil and macrophage numbers and submucosal glands in mouse lung tissues. We prepared cryostat thin sections (5 µm) from lung tissue of uninfected Cftr KO, Cftr S489X mice, wild type or Cftr -/- /Smpd1 +/- mice. We fixed sections on glass slides with acetone, washed them with PBS-Tween (0.2%) and pre-incubated them with normal goat serum, 1:10 diluted in PBS-Tween (0.2%). Then, we incubated the sections with a 1:50 dilution of monoclonal rat antibodies to mouse neutrophils (clone 7/4) or to macrophages (CD68) (Acris) in PBS-Triton (0.5%) overnight at 4 C. After washing with PBS-Tween, (0.1%) we incubated the sections with Cy2-labeled goat antibodies against mouse IgM for 1 h at room temperature, followed by three washing steps with PBS-Tween (0.1%) and an incubation with 2 µg/ml of DAPI (Roche) in PBS-Tween (0.1%) at room temperature. The sections were washed and embedded with mounting medium. For quantitative determination of cell numbers, we examined five sections per mouse lung. From every section, we took 6 to 10 digitalized images (magnification: 200x; area: 850 x 650 µm) and analysed the sections with an Axioplan microscope (Zeiss) using Axiovision (Zeiss). We determined submucosal glands in mouse lungs by staining with the periodic acid Schiff (PAS) reagent. References 1. Snouwaert, J.N. et al. An animal model for cystic fibrosis made by gene targeting. Science 257, (1992). 2. Zhou, L. et al. Correction of lethal intestinal defect in a mouse model of cystic fibrosis by human CFTR. Science 266, (1994). 3. Charizopoulou, N. et al. Instability of the insertional mutation in CftrTgH(neoim)Hgu

15 cystic fibrosis mouse model. BMC Genet. 5, 6 (2004). 4. Charizopoulou N, et al. Spontaneous rescue from cystic fibrosis in a mouse model. BMC Genet. 7, 18 (2006). 5. Dorin, J.R. et al. Cystic fibrosis in the mouse by targeted insertional mutagenesis. Nature 359, (1992). 6. Dorin JR, et al. Long-term survival of the exon 10 insertional cystic fibrosis mutant mouse is a consequence of low level residual wild type Cftr gene expression. Mammalian Genome 5, (1994). 7. van Heeckeren, A.M., Schluchter, M.D., Xue, W. & Davis, P.B. Response to acute lung infection with mucoid Pseudomonas aeruginosa in cystic fibrosis mice. Am. J. Respir. Crit. Care Med. 173, (2006). 8. Nicklas, W. et al. Recommendations for the health monitoring of rodent and rabbit colonies in breeding and experimental units. Lab. Anim. 36, (2002). 9. Boujaoude, L.C. et al. Cystic fibrosis transmembrane regulator regulates uptake of sphingoid base phosphates and lysophosphatidic acid: modulation of cellular activity of sphingosine 1-phosphate. J. Biol. Chem. 276, (2001).

ASSAY OF SPHINGOMYELINASE ACTIVITY

ASSAY OF SPHINGOMYELINASE ACTIVITY ASSAY OF SPHINGOMYELINASE ACTIVITY Protocol for Protein Extraction Stock Solution 1. Leupeptin/hydrochloride (FW 463.0,

More information

Human Apolipoprotein A1 EIA Kit

Human Apolipoprotein A1 EIA Kit A helping hand for your research Product Manual Human Apolipoprotein A1 EIA Kit Catalog Number: 83901 96 assays 1 Table of Content Product Description 3 Assay Principle 3 Kit Components 3 Storage 4 Reagent

More information

SensoLyte pnpp Alkaline Phosphatase Assay Kit *Colorimetric*

SensoLyte pnpp Alkaline Phosphatase Assay Kit *Colorimetric* SensoLyte pnpp Alkaline Phosphatase Assay Kit *Colorimetric* Catalog # 72146 Kit Size 500 Assays (96-well plate) Optimized Performance: This kit is optimized to detect alkaline phosphatase activity Enhanced

More information

Total Phosphatidic Acid Assay Kit

Total Phosphatidic Acid Assay Kit Product Manual Total Phosphatidic Acid Assay Kit Catalog Number MET- 5019 100 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction Phosphatidic Acid (PA) is a critical precursor

More information

Chromatin IP (Isw2) Fix soln: 11% formaldehyde, 0.1 M NaCl, 1 mm EDTA, 50 mm Hepes-KOH ph 7.6. Freshly prepared. Do not store in glass bottles.

Chromatin IP (Isw2) Fix soln: 11% formaldehyde, 0.1 M NaCl, 1 mm EDTA, 50 mm Hepes-KOH ph 7.6. Freshly prepared. Do not store in glass bottles. Chromatin IP (Isw2) 7/01 Toshi last update: 06/15 Reagents Fix soln: 11% formaldehyde, 0.1 M NaCl, 1 mm EDTA, 50 mm Hepes-KOH ph 7.6. Freshly prepared. Do not store in glass bottles. 2.5 M glycine. TBS:

More information

Mitochondrial Trifunctional Protein (TFP) Protein Quantity Microplate Assay Kit

Mitochondrial Trifunctional Protein (TFP) Protein Quantity Microplate Assay Kit PROTOCOL Mitochondrial Trifunctional Protein (TFP) Protein Quantity Microplate Assay Kit DESCRIPTION Mitochondrial Trifunctional Protein (TFP) Protein Quantity Microplate Assay Kit Sufficient materials

More information

Biological Consulting Services

Biological Consulting Services Biological Consulting Services of North Florida/ Inc. May 13, 2009 Aphex BioCleanse Systems, Inc. Dear Sirs, We have completed antimicrobial efficacy study on the supplied Multi-Purpose Solution. The testing

More information

Global Histone H3 Acetylation Assay Kit

Global Histone H3 Acetylation Assay Kit Global Histone H3 Acetylation Assay Kit Catalog Number KA0633 96 assays Version: 06 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Intended Use... 3 Background... 3 Principle

More information

CHAPTER 4 IMMUNOLOGICAL TECHNIQUES

CHAPTER 4 IMMUNOLOGICAL TECHNIQUES CHAPTER 4 IMMUNOLOGICAL TECHNIQUES Nitroblue Tetrazolium Chloride (NBT) Reduction test NBT reduction test was evaluated by employing the method described by Hudson and Hay,1989 based upon principle that

More information

20X Buffer (Tube1) 96-well microplate (12 strips) 1

20X Buffer (Tube1) 96-well microplate (12 strips) 1 PROTOCOL MitoProfile Rapid Microplate Assay Kit for PDH Activity and Quantity (Combines Kit MSP18 & MSP19) 1850 Millrace Drive, Suite 3A Eugene, Oregon 97403 MSP20 Rev.1 DESCRIPTION MitoProfile Rapid Microplate

More information

EPIGENTEK. EpiQuik Global Histone H4 Acetylation Assay Kit. Base Catalog # P-4009 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE

EPIGENTEK. EpiQuik Global Histone H4 Acetylation Assay Kit. Base Catalog # P-4009 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE EpiQuik Global Histone H4 Acetylation Assay Kit Base Catalog # PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE The EpiQuik Global Histone H4 Acetylation Assay Kit is suitable for specifically measuring global

More information

Total Histone H3 Acetylation Detection Fast Kit (Colorimetric)

Total Histone H3 Acetylation Detection Fast Kit (Colorimetric) Total Histone H3 Acetylation Detection Fast Kit (Colorimetric) Catalog Number KA1538 48 assays Version: 02 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Intended Use...

More information

ab65336 Triglyceride Quantification Assay Kit (Colorimetric/ Fluorometric)

ab65336 Triglyceride Quantification Assay Kit (Colorimetric/ Fluorometric) Version 10 Last updated 19 December 2017 ab65336 Triglyceride Quantification Assay Kit (Colorimetric/ Fluorometric) For the measurement of triglycerides in various samples. This product is for research

More information

Chromatin Immunoprecipitation (ChIPs) Protocol (Mirmira Lab)

Chromatin Immunoprecipitation (ChIPs) Protocol (Mirmira Lab) Chromatin Immunoprecipitation (ChIPs) Protocol (Mirmira Lab) Updated 12/3/02 Reagents: ChIP sonication Buffer (1% Triton X-100, 0.1% Deoxycholate, 50 mm Tris 8.1, 150 mm NaCl, 5 mm EDTA): 10 ml 10 % Triton

More information

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION Supplementary Figures Supplementary Figure S1. Binding of full-length OGT and deletion mutants to PIP strips (Echelon Biosciences). Supplementary Figure S2. Binding of the OGT (919-1036) fragments with

More information

EXPERIMENT 13: Isolation and Characterization of Erythrocyte

EXPERIMENT 13: Isolation and Characterization of Erythrocyte EXPERIMENT 13: Isolation and Characterization of Erythrocyte Day 1: Isolation of Erythrocyte Steps 1 through 6 of the Switzer & Garrity protocol (pages 220-221) have been performed by the TA. We will be

More information

2-Deoxyglucose Assay Kit (Colorimetric)

2-Deoxyglucose Assay Kit (Colorimetric) 2-Deoxyglucose Assay Kit (Colorimetric) Catalog Number KA3753 100 assays Version: 01 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Background... 3 General Information...

More information

Title Revision n date

Title Revision n date A. THIN LAYER CHROMATOGRAPHIC TECHNIQUE (TLC) 1. SCOPE The method describes the identification of hydrocortisone acetate, dexamethasone, betamethasone, betamethasone 17-valerate and triamcinolone acetonide

More information

Trident Membrane Protein Extraction Kit

Trident Membrane Protein Extraction Kit Cat. No. Size Shelf life GTX16373 5/ 20 tests 12 months at the appropriate storage temperatures (see below) Contents Component Storage Amount for 5 tests Amount for 20 tests Buffer A -20 o C 2.5 ml 10

More information

Western Immunoblotting Preparation of Samples:

Western Immunoblotting Preparation of Samples: Western Immunoblotting Preparation of Samples: Total Protein Extraction from Culture Cells: Take off the medium Wash culture with 1 x PBS 1 ml hot Cell-lysis Solution into T75 flask Scrap out the cells

More information

EpiQuik Total Histone H3 Acetylation Detection Fast Kit (Colorimetric)

EpiQuik Total Histone H3 Acetylation Detection Fast Kit (Colorimetric) EpiQuik Total Histone H3 Acetylation Detection Fast Kit (Colorimetric) Base Catalog # PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE The EpiQuik Total Histone H3 Acetylation Detection Fast Kit (Colorimetric)

More information

PRODUCT: RNAzol BD for Blood May 2014 Catalog No: RB 192 Storage: Store at room temperature

PRODUCT: RNAzol BD for Blood May 2014 Catalog No: RB 192 Storage: Store at room temperature PRODUCT: RNAzol BD for Blood May 2014 Catalog No: RB 192 Storage: Store at room temperature PRODUCT DESCRIPTION. RNAzol BD is a reagent for isolation of total RNA from whole blood, plasma or serum of human

More information

Lecithin Cholesterol Acyltransferase (LCAT) ELISA Kit

Lecithin Cholesterol Acyltransferase (LCAT) ELISA Kit Product Manual Lecithin Cholesterol Acyltransferase (LCAT) ELISA Kit Catalog Number STA-616 96 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction Cholesterol is a lipid sterol

More information

EPIGENTEK. EpiQuik Global Histone H3 Acetylation Assay Kit. Base Catalog # P-4008 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE

EPIGENTEK. EpiQuik Global Histone H3 Acetylation Assay Kit. Base Catalog # P-4008 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE EpiQuik Global Histone H3 Acetylation Assay Kit Base Catalog # PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE The EpiQuik Global Histone H3 Acetylation Assay Kit is suitable for specifically measuring global

More information

Anti-ceramide Antibody Prevents the Radiation GI Syndrome in Mice

Anti-ceramide Antibody Prevents the Radiation GI Syndrome in Mice Anti-ceramide Antibody Prevents the Radiation GI Syndrome in Mice Jimmy A. Rotolo 1, Branka Stancevic 1, Jianjun Zhang 1, Guoqiang Hua 1, John Fuller 1, Xianglei Yin 1, Adriana Haimovitz-Friedman 2, Kisu

More information

STORE AT 4 o C Version 3

STORE AT 4 o C Version 3 Instruction Manual Advanced Protein Assay Reagent (Cat. # ADV01) ORDERING INFORMATION To order by phone: (303) - 322-2254 To order by Fax: (303) - 322-2257 To order by e-mail: cserve@cytoskeleton.com Technical

More information

Supplementary Information. Sonorensin: A new bacteriocin with potential of an anti-biofilm agent and a food

Supplementary Information. Sonorensin: A new bacteriocin with potential of an anti-biofilm agent and a food Supplementary Information Sonorensin: A new bacteriocin with potential of an anti-biofilm agent and a food biopreservative Lipsy Chopra, Gurdeep Singh, Kautilya Kumar Jena and Debendra K. Sahoo* Biochemical

More information

Alkaline Phosphatase Assay Kit

Alkaline Phosphatase Assay Kit Alkaline Phosphatase Assay Kit Catalog Number KA0817 500 assays Version: 02 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Background... 3 General Information... 4 Materials

More information

Mitochondrial DNA Isolation Kit

Mitochondrial DNA Isolation Kit Mitochondrial DNA Isolation Kit Catalog Number KA0895 50 assays Version: 01 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Background... 3 General Information... 4 Materials

More information

SUPPLEMENT. Materials and methods

SUPPLEMENT. Materials and methods SUPPLEMENT Materials and methods Cell culture and reagents Cell media and reagents were from Invitrogen unless otherwise indicated. Antibiotics and Tet-certified serum were from Clontech. In experiments

More information

Cells and viruses. Human isolates (A/Kawasaki/173/01 [H1N1], A/Yokohama/2057/03 [H3N2],

Cells and viruses. Human isolates (A/Kawasaki/173/01 [H1N1], A/Yokohama/2057/03 [H3N2], Supplementary information Methods Cells and viruses. Human isolates (A/Kawasaki/173/01 [H1N1], A/Yokohama/2057/03 [H3N2], and A/Hong Kong/213/03 [H5N1]) were grown in Madin-Darby canine kidney (MDCK) cells

More information

Luminescent platforms for monitoring changes in the solubility of amylin and huntingtin in living cells

Luminescent platforms for monitoring changes in the solubility of amylin and huntingtin in living cells Electronic Supplementary Material (ESI) for Molecular BioSystems. This journal is The Royal Society of Chemistry 2016 Contents Supporting Information Luminescent platforms for monitoring changes in the

More information

2,6,9-Triazabicyclo[3.3.1]nonanes as overlooked. amino-modification products by acrolein

2,6,9-Triazabicyclo[3.3.1]nonanes as overlooked. amino-modification products by acrolein Supplementary Information 2,6,9-Triazabicyclo[3.3.1]nonanes as overlooked amino-modification products by acrolein Ayumi Tsutsui and Katsunori Tanaka* Biofunctional Synthetic Chemistry Laboratory, RIKEN

More information

Alkaline Phosphatase Assay Kit (Fluorometric)

Alkaline Phosphatase Assay Kit (Fluorometric) Alkaline Phosphatase Assay Kit (Fluorometric) Catalog Number KA0820 500 assays Version: 02 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Background... 3 General Information...

More information

EPIGENTEK. EpiQuik Global Acetyl Histone H3K27 Quantification Kit (Colorimetric) Base Catalog # P-4059 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE

EPIGENTEK. EpiQuik Global Acetyl Histone H3K27 Quantification Kit (Colorimetric) Base Catalog # P-4059 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE EpiQuik Global Acetyl Histone H3K27 Quantification Kit (Colorimetric) Base Catalog # P-4059 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE The EpiQuik Global Acetyl Histone H3K27 Quantification Kit (Colorimetric)

More information

Mammalian Membrane Protein Extraction Kit

Mammalian Membrane Protein Extraction Kit Mammalian Membrane Protein Extraction Kit Catalog number: AR0155 Boster s Mammalian Membrane Protein Extraction Kit is a simple, rapid and reproducible method to prepare cellular protein fractions highly

More information

Gladstone Institutes, University of California (UCSF), San Francisco, USA

Gladstone Institutes, University of California (UCSF), San Francisco, USA Fluorescence-linked Antigen Quantification (FLAQ) Assay for Fast Quantification of HIV-1 p24 Gag Marianne Gesner, Mekhala Maiti, Robert Grant and Marielle Cavrois * Gladstone Institutes, University of

More information

Manual. Precision Red Advanced Protein Assay Reagent. Cat. # ADV02. cytoskeleton.com. Cytoskeleton, Inc.

Manual. Precision Red Advanced Protein Assay Reagent. Cat. # ADV02. cytoskeleton.com. Cytoskeleton, Inc. The Protein Experts Manual Cytoskeleton, Inc. V. 6.0 Precision Red Advanced Protein Assay Reagent Cat. # ADV02 cytoskeleton.com Phone: (303) 322.2254 Fax: (303) 322.2257 Customer Service: cserve@cytoskeleton.com

More information

9( )- Hydroxyoctadecadienoic Acid ELISA

9( )- Hydroxyoctadecadienoic Acid ELISA Package Insert 9( )- Hydroxyoctadecadienoic Acid ELISA 96 Wells For Research Use Only v. 1.0 Eagle Biosciences, Inc. 82 Broad Street, Suite 383, Boston, MA 02110 Phone: 866-419-2019 Fax: 617-419-1110 INTRODUCTION

More information

Work-flow: protein sample preparation Precipitation methods Removal of interfering substances Specific examples:

Work-flow: protein sample preparation Precipitation methods Removal of interfering substances Specific examples: Dr. Sanjeeva Srivastava IIT Bombay Work-flow: protein sample preparation Precipitation methods Removal of interfering substances Specific examples: Sample preparation for serum proteome analysis Sample

More information

Protocol for Gene Transfection & Western Blotting

Protocol for Gene Transfection & Western Blotting The schedule and the manual of basic techniques for cell culture Advanced Protocol for Gene Transfection & Western Blotting Schedule Day 1 26/07/2008 Transfection Day 3 28/07/2008 Cell lysis Immunoprecipitation

More information

(A) PCR primers (arrows) designed to distinguish wild type (P1+P2), targeted (P1+P2) and excised (P1+P3)14-

(A) PCR primers (arrows) designed to distinguish wild type (P1+P2), targeted (P1+P2) and excised (P1+P3)14- 1 Supplemental Figure Legends Figure S1. Mammary tumors of ErbB2 KI mice with 14-3-3σ ablation have elevated ErbB2 transcript levels and cell proliferation (A) PCR primers (arrows) designed to distinguish

More information

High-density Lipoprotein Cholesterol (HDL-C) Assay Kit

High-density Lipoprotein Cholesterol (HDL-C) Assay Kit (FOR RESEARCH USE ONLY. DO NOT USE IT IN CLINICAL DIAGNOSIS!) High-density Lipoprotein Cholesterol (HDL-C) Assay Kit (Double reagents) Catalog No: E-BC-K221 Method: Colorimetric method Specification: 96T

More information

Biodegradable Zwitterionic Nanogels with Long. Circulation for Antitumor Drug Delivery

Biodegradable Zwitterionic Nanogels with Long. Circulation for Antitumor Drug Delivery Supporting Information Biodegradable Zwitterionic Nanogels with Long Circulation for Antitumor Drug Delivery Yongzhi Men, Shaojun Peng, Peng Yang, Qin Jiang, Yanhui Zhang, Bin Shen, Pin Dong, *, Zhiqing

More information

AMYLOGLUCOSIDASE from ASPERGILLUS NIGER, var.

AMYLOGLUCOSIDASE from ASPERGILLUS NIGER, var. AMYLOGLUCOSIDASE from ASPERGILLUS NIGER, var. SYNONYMS INS No. 1100 Prepared at the 59 th JECFA (2002) and published in FNP 52 Add 10 (2002), superseding tentative specifications prepared at the 55 th

More information

DEVELOPMENTAL VALIDATION OF SPERM HY-LITER EXPRESS Jennifer Old, Chris Martersteck, Anna Kalinina, Independent Forensics, Lombard IL

DEVELOPMENTAL VALIDATION OF SPERM HY-LITER EXPRESS Jennifer Old, Chris Martersteck, Anna Kalinina, Independent Forensics, Lombard IL DEVELOPMENTAL VALIDATION OF SPERM HY-LITER EXPRESS Jennifer Old, Chris Martersteck, Anna Kalinina, Independent Forensics, Lombard IL Background and Introduction SPERM HY-LITER is the first immunofluorescent

More information

ab Human Citrate Synthase (CS) Activity Assay Kit

ab Human Citrate Synthase (CS) Activity Assay Kit ab119692 Human Citrate Synthase (CS) Activity Assay Kit Instructions for Use For the measurement of mitochondrial citrate synthase (CS) activity in Human samples This product is for research use only and

More information

Nature Protocols: doi: /nprot Supplementary Figure 1. Fluorescent titration of probe CPDSA.

Nature Protocols: doi: /nprot Supplementary Figure 1. Fluorescent titration of probe CPDSA. Supplementary Figure 1 Fluorescent titration of probe CPDSA. Fluorescent titration of probe CPDSA (10 um) upon addition of GSH in HEPES (10 mm, ph = 7.4) containing 10% DMSO. Each spectrum was recorded

More information

DAG (Diacylglycerol) Assay Kit

DAG (Diacylglycerol) Assay Kit Product Manual DAG (Diacylglycerol) Assay Kit Catalog Number MET-5028 100 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction Diacylglycerols (DAG) are key intermediates in the

More information

Whole Mount Drosophila Embryo In Situ Hybridization with RNA probes 2/5/2001 Leslie Vosshall

Whole Mount Drosophila Embryo In Situ Hybridization with RNA probes 2/5/2001 Leslie Vosshall Whole Mount Drosophila Embryo In Situ Hybridization with RNA probes 2/5/2001 Leslie Vosshall DAY ONE All incubations are done at room temperature unless otherwise noted. All solutions and all containers

More information

Evaluation of directed and random motility in microslides Assessment of leukocyte adhesion in flow chambers

Evaluation of directed and random motility in microslides Assessment of leukocyte adhesion in flow chambers Evaluation of directed and random motility in microslides Motility experiments in IBIDI microslides, image acquisition and processing were performed as described. PMN, which ended up in an angle < 180

More information

PREPARATION OF IF- ENRICHED CYTOSKELETAL PROTEINS

PREPARATION OF IF- ENRICHED CYTOSKELETAL PROTEINS TMM,5-2011 PREPARATION OF IF- ENRICHED CYTOSKELETAL PROTEINS Ice-cold means cooled in ice water. In order to prevent proteolysis, make sure to perform all steps on ice. Pre-cool glass homogenizers, buffers

More information

ab Glucose Uptake Assay Kit (colorimetric) 1

ab Glucose Uptake Assay Kit (colorimetric) 1 Version 16 Last updated 10 January 2018 ab136955 Glucose Uptake Assay Kit (Colorimetric) For the measurement of Glucose uptake in a variety of cells. This product is for research use only and is not intended

More information

VEGFR2-Mediated Vascular Dilation as a Mechanism of VEGF-Induced Anemia and Bone Marrow Cell Mobilization

VEGFR2-Mediated Vascular Dilation as a Mechanism of VEGF-Induced Anemia and Bone Marrow Cell Mobilization Cell Reports, Volume 9 Supplemental Information VEGFR2-Mediated Vascular Dilation as a Mechanism of VEGF-Induced Anemia and Bone Marrow Cell Mobilization Sharon Lim, Yin Zhang, Danfang Zhang, Fang Chen,

More information

Ph. Eur. Reference Standard - LEAFLET

Ph. Eur. Reference Standard - LEAFLET European Directorate for the Quality of Medicines & HealthCare European Pharmacopoeia (Ph. Eur.) 7, Allée Kastner CS 30026, F-67081 Strasbourg (France) Tel. +33 (0)3 88 41 20 35 Fax. + 33 (0)3 88 41 27

More information

In vitro bactericidal assay Fig. S8 Gentamicin protection assay Phagocytosis assay

In vitro bactericidal assay Fig. S8 Gentamicin protection assay Phagocytosis assay In vitro bactericidal assay Mouse bone marrow was isolated from the femur and the tibia. Cells were suspended in phosphate buffered saline containing.5% BSA and 2 mm EDTA and filtered through a cell strainer.

More information

AMPK Assay. Require: Sigma (1L, $18.30) A4206 Aluminum foil

AMPK Assay. Require: Sigma (1L, $18.30) A4206 Aluminum foil AMPK Assay Require: Acetone Sigma (1L, $18.30) A4206 Aluminum foil Ammonium sulfate Fisher BP212R-1 AMP Sigma A1752 ATP Sigma A6144 (alt. use A7699) Beta-mercaptoethanol Sigma M6250 (alt. use M7154) Bio-Rad

More information

The Schedule and the Manual of Basic Techniques for Cell Culture

The Schedule and the Manual of Basic Techniques for Cell Culture The Schedule and the Manual of Basic Techniques for Cell Culture 1 Materials Calcium Phosphate Transfection Kit: Invitrogen Cat.No.K2780-01 Falcon tube (Cat No.35-2054:12 x 75 mm, 5 ml tube) Cell: 293

More information

Instructions for Use. APO-AB Annexin V-Biotin Apoptosis Detection Kit 100 tests

Instructions for Use. APO-AB Annexin V-Biotin Apoptosis Detection Kit 100 tests 3URGXFW,QIRUPDWLRQ Sigma TACS Annexin V Apoptosis Detection Kits Instructions for Use APO-AB Annexin V-Biotin Apoptosis Detection Kit 100 tests For Research Use Only. Not for use in diagnostic procedures.

More information

GB Translated English of Chinese Standard: GB NATIONAL STANDARD OF THE

GB Translated English of Chinese Standard: GB NATIONAL STANDARD OF THE Translated English of Chinese Standard: GB4789.30-2016 www.chinesestandard.net Buy True-PDF Auto-delivery. Sales@ChineseStandard.net GB NATIONAL STANDARD OF THE PEOPLE S REPUBLIC OF CHINA GB 4789.30-2016

More information

(Stratagene, La Jolla, CA) (Supplemental Fig. 1A). A 5.4-kb EcoRI fragment

(Stratagene, La Jolla, CA) (Supplemental Fig. 1A). A 5.4-kb EcoRI fragment SUPPLEMENTAL INFORMATION Supplemental Methods Generation of RyR2-S2808D Mice Murine genomic RyR2 clones were isolated from a 129/SvEvTacfBR λ-phage library (Stratagene, La Jolla, CA) (Supplemental Fig.

More information

Trypsin Mass Spectrometry Grade

Trypsin Mass Spectrometry Grade 058PR-03 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name Trypsin Mass Spectrometry Grade A Chemically Modified, TPCK treated, Affinity Purified

More information

SUPPLEMENTARY MATERIAL. Sample preparation for light microscopy

SUPPLEMENTARY MATERIAL. Sample preparation for light microscopy SUPPLEMENTARY MATERIAL Sample preparation for light microscopy To characterize the granulocytes and melanomacrophage centers, cross sections were prepared for light microscopy, as described in Material

More information

For the quantitative measurement of ATP Synthase Specific activity in samples from Human, Rat and Cow

For the quantitative measurement of ATP Synthase Specific activity in samples from Human, Rat and Cow ab109716 ATP Synthase Specific Activity Microplate Assay Kit Instructions for Use For the quantitative measurement of ATP Synthase Specific activity in samples from Human, Rat and Cow This product is for

More information

Supplementary Figures

Supplementary Figures Inhibition of Pulmonary Anti Bacterial Defense by IFN γ During Recovery from Influenza Infection By Keer Sun and Dennis W. Metzger Supplementary Figures d a Ly6G Percentage survival f 1 75 5 1 25 1 5 1

More information

Appendix A: Preparation of Media and Chemicals. Malt Extract Agar (MEA) weighing g was dissolved in 400 ml of distilled water

Appendix A: Preparation of Media and Chemicals. Malt Extract Agar (MEA) weighing g was dissolved in 400 ml of distilled water Appendix A: Preparation of Media and Chemicals Preparation of Malt Extract Agar (MEA) Malt Extract Agar (MEA) weighing 13.44 g was dissolved in 400 ml of distilled water in an Erlenmeyer flask using a

More information

Supplementary Materials and Methods

Supplementary Materials and Methods Supplementary Materials and Methods Whole Mount X-Gal Staining Whole tissues were collected, rinsed with PBS and fixed with 4% PFA. Tissues were then rinsed in rinse buffer (100 mm Sodium Phosphate ph

More information

SUPPLEMENTARY INFORMATION. Bacterial strains and growth conditions. Streptococcus pneumoniae strain R36A was

SUPPLEMENTARY INFORMATION. Bacterial strains and growth conditions. Streptococcus pneumoniae strain R36A was SUPPLEMENTARY INFORMATION Bacterial strains and growth conditions. Streptococcus pneumoniae strain R36A was grown in a casein-based semisynthetic medium (C+Y) supplemented with yeast extract (1 mg/ml of

More information

Figure S1. PMVs from THP-1 cells expose phosphatidylserine and carry actin. A) Flow

Figure S1. PMVs from THP-1 cells expose phosphatidylserine and carry actin. A) Flow SUPPLEMENTARY DATA Supplementary Figure Legends Figure S1. PMVs from THP-1 cells expose phosphatidylserine and carry actin. A) Flow cytometry analysis of PMVs labelled with annexin-v-pe (Guava technologies)

More information

Lipase Assay Kit. Catalog Number KA assays Version: 02. Intended for research use only.

Lipase Assay Kit. Catalog Number KA assays Version: 02. Intended for research use only. Lipase Assay Kit Catalog Number KA1654 100 assays Version: 02 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Intended Use... 3 Principle of the Assay... 3 General Information...

More information

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION FOR Liver X Receptor α mediates hepatic triglyceride accumulation through upregulation of G0/G1 Switch Gene 2 (G0S2) expression I: SUPPLEMENTARY METHODS II: SUPPLEMENTARY FIGURES

More information

Glutathione S-Transferase Assay Kit

Glutathione S-Transferase Assay Kit Glutathione S-Transferase Assay Kit Catalog Number KA1316 96 assays Version: 05 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Background... 3 Principle of the Assay...

More information

Phospholipid Assay Kit

Phospholipid Assay Kit Phospholipid Assay Kit Catalog Number KA1635 100 assays Version: 06 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Intended Use... 3 Background... 3 General Information...

More information

2-Deoxyglucose (2DG) Uptake Measurement kit

2-Deoxyglucose (2DG) Uptake Measurement kit Document#:K2DG13516E For research use only. Not for clinical diagnosis. Catalog No. CSR-OKP-PMG-K1E 2-Deoxyglucose (2DG) Uptake Measurement kit Introduction Measurement of 2-deoxyglucose (2DG) uptake in

More information

SUPPLEMENTAL MATERIAL. Supplementary Methods

SUPPLEMENTAL MATERIAL. Supplementary Methods SUPPLEMENTAL MATERIAL Supplementary Methods Culture of cardiomyocytes, fibroblasts and cardiac microvascular endothelial cells The isolation and culturing of neonatal rat ventricular cardiomyocytes was

More information

Glycosyltransferase Activity Kit

Glycosyltransferase Activity Kit Glycosyltransferase Activity Kit Catalog Number EA001 This package insert must be read in its entirety before using this product. For research use only. Not for use in diagnostic procedures. TABLE OF CONTENTS

More information

OxiSelect Hydrogen Peroxide Assay Kit (Colorimetric)

OxiSelect Hydrogen Peroxide Assay Kit (Colorimetric) Product Manual OxiSelect Hydrogen Peroxide Assay Kit (Colorimetric) Catalog Number STA-343 5 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction Oxidative stress is a physiological

More information

Glycerol- 3- Phosphate (G3P) Assay Kit (Colorimetric)

Glycerol- 3- Phosphate (G3P) Assay Kit (Colorimetric) Product Manual Glycerol- 3- Phosphate (G3P) Assay Kit (Colorimetric) Catalog Number MET- 5075 100 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction Glycerol-3-phosphate (G3P)

More information

22 Bicozamycin (Bicyclomycin)

22 Bicozamycin (Bicyclomycin) 22 Bicozamycin (Bicyclomycin) OH O H N O O OH HO [Summary of bicozamycin] C 12 H 18 N 2 O 7 MW: 302.3 CAS No.: 38129-37-2 Bicozamycin (BZM) is an antibiotic obtained from a fermented culture of Streptomyces

More information

Phospholipid Assay Kit

Phospholipid Assay Kit Phospholipid Assay Kit Catalog Number KA1635 100 assays Version: 05 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Intended Use... 3 Background... 3 General Information...

More information

Calibration Protocols

Calibration Protocols Calibration Protocols (1)OD 600 Reference point LUDOX Protocol Materials: 1ml LUDOX CL-X (provided in kit) ddh20 (provided by team) 96 well plate, black with clear flat bottom preferred (provided by team)

More information

The following protocol describes the isolation of nuclei from tissue. Item. Catalog No Manufacturer

The following protocol describes the isolation of nuclei from tissue. Item. Catalog No Manufacturer SOP: Nuclei isolation from tissue and DNaseI treatment Date modified: 090923 Modified by: P. Sabo. (UW) The following protocol describes the isolation of nuclei from tissue. Ordering Information Item.

More information

ab CytoPainter Golgi/ER Staining Kit

ab CytoPainter Golgi/ER Staining Kit ab139485 CytoPainter Golgi/ER Staining Kit Instructions for Use Designed to detect Golgi bodies and endoplasmic reticulum by microscopy This product is for research use only and is not intended for diagnostic

More information

Supporting Information File S2

Supporting Information File S2 Pulli et al. Measuring Myeloperoxidase Activity in Biological Samples Page 1 of 6 Supporting Information File S2 Step-by-Step Protocol Reagents: Sucrose (Sigma, S3089) CaCl 2 (Sigma, C5770) Heparin Sodium

More information

B16-F10 (Mus musculus skin melanoma), NCI-H460 (human non-small cell lung cancer

B16-F10 (Mus musculus skin melanoma), NCI-H460 (human non-small cell lung cancer Electronic Supplementary Material (ESI) for ChemComm. This journal is The Royal Society of Chemistry 2017 Experimental Methods Cell culture B16-F10 (Mus musculus skin melanoma), NCI-H460 (human non-small

More information

ab Complex II Enzyme Activity Microplate Assay Kit

ab Complex II Enzyme Activity Microplate Assay Kit ab109908 Complex II Enzyme Activity Microplate Assay Kit Instructions for Use For the quantitative measurement of Complex II activity in samples from Human, Rat, Mouse and Cow This product is for research

More information

SensoLyte 520 Cathepsin K Assay Kit *Fluorimetric*

SensoLyte 520 Cathepsin K Assay Kit *Fluorimetric* SensoLyte 520 Cathepsin K Assay Kit *Fluorimetric* Catalog # 72171 Kit Size 100 Assays (96-well plate) Optimized Performance: This kit is optimized to detect Cathepsin K activity. Enhanced Value: Ample

More information

Recipes for Media and Solution Preparation SC-ura/Glucose Agar Dishes (20mL/dish, enough for 8 clones)

Recipes for Media and Solution Preparation SC-ura/Glucose Agar Dishes (20mL/dish, enough for 8 clones) Protocol: 300 ml Yeast culture preparation Equipment and Reagents needed: Autoclaved toothpicks Shaker Incubator set at 30 C Incubator set at 30 C 60 mm 2 sterile petri dishes Autoclaved glass test tubes

More information

TRACP & ALP Assay Kit

TRACP & ALP Assay Kit Cat. # MK301 For Research Use TRACP & ALP Assay Kit Product Manual Table of Contents I. Description...3 II. III. IV. Introduction...3 Components...4 Materials Required but not Provided...4 V. Storage...4

More information

ab ATP Synthase Enzyme Activity Microplate Assay Kit

ab ATP Synthase Enzyme Activity Microplate Assay Kit ab109714 ATP Synthase Enzyme Activity Microplate Assay Kit Instructions for Use For the quantitative measurement of ATP Synthase activity in samples from Human, Rat and Cow This product is for research

More information

Ph. Eur. Reference Standard - LEAFLET

Ph. Eur. Reference Standard - LEAFLET European Directorate for the Quality of Medicines & HealthCare European Pharmacopoeia (Ph. Eur.) 7, Allée Kastner CS 30026, F-67081 Strasbourg (France) Tel. +33 (0)3 88 41 20 35 Fax. + 33 (0)3 88 41 27

More information

supplementary information

supplementary information Figure S1 Nucleotide binding status of RagA mutants. Wild type and mutant forms of MycRagA was transfected into HEK293 cells and the transfected cells were labeled with 32 Pphosphate. MycRagA was immunoprecipitated

More information

ab Lipid Peroxidation (MDA) Assay kit (Colorimetric/ Fluorometric)

ab Lipid Peroxidation (MDA) Assay kit (Colorimetric/ Fluorometric) Version 10b Last updated 19 December 2018 ab118970 Lipid Peroxidation (MDA) Assay kit (Colorimetric/ Fluorometric) For the measurement of Lipid Peroxidation in plasma, cell culture and tissue extracts.

More information

Plasma Membrane Protein Extraction Kit

Plasma Membrane Protein Extraction Kit ab65400 Plasma Membrane Protein Extraction Kit Instructions for Use For the rapid and sensitive extraction and purification of Plasma Membrane proteins from cultured cells and tissue samples. This product

More information

Cathepsin K Activity Assay Kit

Cathepsin K Activity Assay Kit Cathepsin K Activity Assay Kit Catalog Number KA0769 100 assays Version: 03 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Background... 3 General Information... 4 Materials

More information

Detailed step-by-step operating procedures for NK cell and CTL degranulation assays

Detailed step-by-step operating procedures for NK cell and CTL degranulation assays Supplemental methods Detailed step-by-step operating procedures for NK cell and CTL degranulation assays Materials PBMC isolated from patients, relatives and healthy donors as control K562 cells (ATCC,

More information

Essential Medium, containing 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin. Huvec were cultured in

Essential Medium, containing 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin. Huvec were cultured in Supplemental data Methods Cell culture media formulations A-431 and U-87 MG cells were maintained in Dulbecco s Modified Eagle s Medium. FaDu cells were cultured in Eagle's Minimum Essential Medium, containing

More information