LentiBoost Lentiviral Transduction Enhancer

Transcription

1 Product Manual Lot number: IP1108_LM_R_5 LentiBoost Lentiviral Transduction Enhancer SB-P-LV SB-P-LV Catalogue number: 100 standard transductions 300 standard transductions Shipped at room temperature Store at -20 C FOR RESEARCH USE ONLY Not for clinical use

2 PRODUCT DESCRIPTION: LentiBoost has been developed to enhance the uptake of lentiviral vectors into cells and cell lines. It acts by reducing electrostatic repulsion between the virion and cell membranes. LentiBoost is especially recommended for cells, which are hard to transduce or which are sensitive to other transduction enhancers (e.g. Polybrene). Procedure LentiBoost consists of 2 solutions. In a first step the best ratio and concentration of both solutions has to be determined in order to get highest transduction efficiency as well as lowest toxic effects. If suitable conditions for lentiviral transduction of your cell line of interest are unknown we recommend to test at least 2 different multiplicities of infection (MOI) based on infectious titer, namely MOI 2 and 10 using a lentiviral vector encoding a fluorescent protein. Lentiviral transduction with LentiBoost can be performed either according to the standard protocol or the centrifugation protocol. We recommend to begin with the standard procedure. If the transduction efficiency turns out to be too low you may get better results using the centrifugation protocol. Short overview: Standard protocol: Cells are seeded the day before transduction in the appropriate format. Prior to transduction mixtures of 17 different ratios of solution A and solution B, lentivirus and cell culture medium are prepared according to table 2. Medium has to be aspirated from the cells and replaced by the LentiBoost mixes. The following day medium has to be changed to normal growth medium. Transduction efficiency should be determined 4-6 days after transduction via fluorescent microscopy or FACS analysis. Centifugation protocol: For hard to transduce cells we extended the standard procedure by a centrifugation step. Please be aware of the fact that additional centrifugation usually results in lower cell viability. For the centrifugation protocol cells have to be detached and counted at the day of transduction. Cells are pelleted and resuspended in the Lentivirus/LentiBoost mixes, prepared according to standard procedure. After a centrifugation step at 1000g for 30 min cells are seeded in the appropriate wells and incubated at standard culture conditions. Culture medium is changed the next day. Transduction efficiency should be determined 4-6 days after transduction e.g. via fluorescent microscopy or FACS analysis. MATERIALS SUPPLIED: - LentiBoost: SB-P-LV vial à 500 µl LentiBoost 100x Solution A (sufficient for 100 transductions in 24well) 1 vial à 500 µl LentiBoost 100x Solution B (sufficient for 100 transductions in 24well) SB-P-LV vial à 1500 µl LentiBoost 100x Solution A (sufficient for 300 transductions in 24well) 1 vial à 1500 µl LentiBoost 100x Solution B (sufficient for 300 transductions in 24well) MATERIALS NOT SUPPLIED: - Cells - Cell culture growth medium - 37 C incubator - Lentivirus STORAGE Store at -20 C. Lot# IP1108_LM_R_5 Page 2/6

3 PROTOCOL: STANDARD PROTOCOL Day 1: Seeding cells The day before transduction, seed equal amounts of cells into desired plate format. The number of cells to be plated per well depends on the cell type. Please refer to table 1 to get an appropriate number of cells per well. 2E+04 cells in 24 well were found to be adequate for most adherent growing cells, e.g. NIH-3T3. Ideally, cells should exhibit about 20% confluence at the time of transduction. Table 1: Recommended cell numbers and volumes to be plated for lentiviral transduction Format Cell Number/Well Volume/well ( l) 96 well 3E well 1E well 2E well 4E well 1E Day 2: Transduction If suitable conditions for lentiviral transduction of your cell line of interest are unknown we recommend to test at least 2 different multiplicities of infection (MOI) based on infectious titer, namely MOI 2 and 10 using a lentiviral vector encoding a fluorescent protein. Thaw virus and dilute virus into complete culture media sufficient for 16 samples per virus concentration (MOI). Aliquot virus/media mastermix (e.g. in 500 µl units for 24 well). Mix by inverting; do not vortex. Add solution A and solution B according to Table 2. You may prepare predilutions in PBS if required. Prepare one vial with medium only (#17) without lentivirus to control viability of lentivirus Mix vials by inverting the tubes (do not vortex) Aspirate medium from cells and apply Virus-Medium-LentiBoost mixes Incubate cells overnight at standard cell culture conditions Day 3: Medium exchange Remove medium from cells and add appropriate amount of normal growth medium (e.g. 500 µl for 24 well plate) Optional: Day 4-5: Maintenance If the cells do not tolerate to be grown at too high confluences they can be split on day 4-5 with the regular splitting ratio. Medium exchange has to be performed at normal intervals. Lot# IP1108_LM_R_5 Page 3/6

4 Day 4-8: Analysis Fluorescent intensities of stably transduced cells have to be monitored daily under the microscope. Usually maximal intensity of fluorescent signals is reached 4-6 days after transduction. Determine LentiBoost conditions that allow for high transduction efficiency and low toxicity Table 2: Recommended setup for an initial transduction experiment withlentiboost LentiBoost Solution/Medium Ratio MOI Solution A Solution B Total Volume (Medium + Virus) Example 24 well Solution A Solution B #1 1:100 1: µl 5 µl 5 µl #2 1: µl 5 µl - #3-1: µl - 5 µl #4 1:2000 1: µl 0,25 µl 0,25 µl #5 1: µl 0,25 µl - #6-1: µl - 0,25 µl #7 1:100 1: µl 5 µl 0,25 µl #8 1:2000 1: µl 0,25 µl 5 µl #9 1:100 1: µl 5 µl 5 µl #10 1: µl 5 µl - #11-1: µl - 5 µl #12 1:2000 1: µl 0,25 µl 0,25 µl #13 1: µl 0,25 µl - #14-1: µl - 0,25 µl #15 1:100 1: µl 5 µl 0,25 µl #16 1:2000 1: µl 0,25 µl 5 µl # µl - - Lot# IP1108_LM_R_5 Page 4/6

5 CENTRIFUGATION PROTOCOL Day 1 Detach cells at the day of transduction and seed them into 17 wells with the appropriate cell number. Refer to table 1 (e.g. 2E+04 for 24 well). Add virus and LentiBoost according to table 2 Centrifuge the whole plate at 800g for 90 min Gently swirl the plate to spread cells homogenously on the plate surface Incubate cells overnight at standard cell culture conditions Day 3: Medium exchange Remove medium from cells and apply appropriate amount of normal growth medium (e.g µl for 24 well plate) Optional: Day 4-5: Maintenance If the cells do not tolerate to be grown at too high confluences they can be split on day 4-5 with the regular splitting ratio. Medium exchange has to be performed at normal intervals. Day 4-8: Analysis Fluorescent intensities of stably transduced cells have to be monitored daily under the microscope. Usually maximal intensity of fluorescent signals is reached 4-6 days after transduction. Determine LentiBoost conditions that allow for high transduction efficiency and low toxicity Lot# IP1108_LM_R_5 Page 5/6

6 RLU LentiBoost DATA: Comparison of luc2 expression 72h after lentiviral transduction using different enhancers no virus no substance 8 µg/ml Polybrene LentiMAX LentiBoost 50 0 plate blank (no cells) Panc-1 NIH/3T3 Figure 1: Comparison of luc2 expression 72h after lentiviral transduction using different enhancers and centrifugation methode. LentiBoost showed in two cell lines higher RLU than Polybrene TROUBLESHOOTING: problem Reason Solution Low transduction efficiency MOI used was too low Infectious viral titer is too low Cells are very difficult to transduce LentiBoost concentration used was too low Low viability MOI used was too high Use less Lentivirus LentiBoost concentration used was too high Cells are sensitive against lentiviral treatment Use higher amount of Lentivirus: Up to MOI100 is possible Check infectious titer via transduction of HEK 293 cells with serial dilutions of your GFPexpressing lentiviral vector. Repeat experiment with correct titer Use centrifugation protocol instead of standard protocol Increase concentrations of Solution A and B Decrease concentrations of Solution A and B Change medium 4h after transduction or directly after centrifugation DISCLAIMER This product is sold for research and development purposes only and is not to be incorporated into products for resale without the permission from SIRION BIOTECH GmbH and may not be given to third parties for commercial use, sale or resale. CONTACT INFORMATION SIRION BIOTECH GmbH Am Klopferspitz Martinsried Germany Phone: info@sirion-biotech.de Lot# IP1108_LM_R_5 Page 6/6