L6 GLUT4myc Cell Growth Protocol Background: Parental L6 cells selected for high fusion (2, 3) were stably transfected with a rat GLUT4 cdna carrying a myc epitope (recognized by the commercially available monoclonal antibody, 9E10) located between codons 66 and 67 in the exofacial loop of the GLUT4 protein (L6-GLUT4myc) as described by Kishi et al. (1). The expression vector carrying the GLUT4myc cdna, pcxn2, has a CMV promoter. Blasticidin resistance was used as a selection marker to create the stable line. Methods for detecting GLUT4myc on the cell surface of intact cells by colourimetric- or immunofluorescence-based assays have been described by Wang et al. (4, 5). Materials: 1. α-minimal Essential Medium (α-mem) Gibco #12571-063 2. Fetal Bovine Serum (FBS) Gibco #26140-038 3. Antibiotic / Antimycotic (AB) Gibco #15240-062 4. Trypsin 0.25% Liquid Gibco #15050-057 5. Dimethyl Sulfoxide (DMSO) Sigma # D-8779 6. Trypan Blue (0.4% in saline) Gibco #15250-020 7. Blasticidin-HCl Calbiochem # 203350 Growth Medium: Differentiation Medium: Freezing Medium: Growing Conditions: α-mem / 10% FBS / 1% AB (v/v) α-mem / 2% FBS / 1% AB (v/v) α-mem / 10% FBS / 1% AB (v/v) / 10% DMSO 5% CO 2 @ 37 C Start-up and Maintenance 1. Thaw ampule of L6 cells rapidly @ 37 C. 2. Carefully resuspend cells using a sterile pipette (3-5 times). 3. Add the cells slowly & directly to temperature pre-equilibrated growth medium in a 75 cm 2 flask, mix gently and place in the incubator. 4. Change the medium (decanting slowly) after 1-2 hours (to remove the DMSO). Thereafter change medium at least every 48 hours. 5. Cells can be maintained for 10-15 passages (5-7 weeks) before a new vial should be started. 6. Before using the cells in experiments expand cells in order to freeze down several vials for future use (see below). 7. This step is recommended. The cells can be maintained in the presence of the 2 µg/ml blasticidin-hcl during the passages. However, upon seeding cells for experiments, eliminate the drug from the culture medium. 8. Periodically (every four months), your lab may want to re-select the L6- GLUT4myc cell line in blasticidin to ensure clonal stability. Use Blasticidin at 10 µg/ml for 3 passages of 1:10 splittings of the cells. Then freeze down the cells (see below) in the absence of blasticidin.
Trypsinization N.B. Trypsinization of cells is done at less at 60-80% confluency to maintain passages & seeding for experiments. It is imperative that the cells are not aligned ( i.e. committed) by this time, otherwise you may be selecting for myoblasts with a non-fusing phenotype. Per Flask: 250 ml (75 cm 2 ) 1. Decant medium and rinse twice with 1X PBS 2. Decant and add 4 ml of trypsin. Observe under a microscope for cell rounding in 2-3 minutes. 3. Decant medium carefully and add 5 ml of growth medium, and dislodge cells by tapping the flask on the sides. Again observe under microscope that the cells have detached. 4. Transfer cells into sterile conical 50 ml centrifuge tube. Rinse flask once with 5ml growth medium and add to the tube. 5. Centrifuge the cells at room temp. for 5 min. @ 800-1000 xg in a desk-top centrifuge 6. Resuspend cells in 5 ml growth medium and count in a haemocytometer chamber. Per Flask: 800 ml (175 cm 2 ) Perform as above, but use 4 ml of trypsin and 10 ml of growth medium to dislodge the cells and 10 ml of growth medium to resuspend the pelleted cells. Counting Cells After trypsinization and the cells are resuspended, they can be counted using a haemocytometer as follows: Directions: Place a drop of cells onto the haemocytometer (Hauser) with the cover glass in place. Count cells in the 4 zones of 16 squares (A, B, C, D). The number of cells in suspension = 1/4 (total cell count) x 10,000 cells/ml. Freezing Down Cells Freeze-down medium: Add DMSO to growth medium to a final concentration of 10 % and then filter the mixture through a 0.2 µm filter. 1 Perform the trypsinization of the cells as described above and resuspend the cells in freeze-down medium to a concentration of 1 X10 6 Cells/ml. E.g. Per Flask: 250 ml (75 cm 2 ) at ~ 80 % confluency: 6 ml Per Flask: 800 ml (175 cm 2 ) at ~ 80 % confluency: 12 ml 2. Transfer 1.5 ml per cryotube. Freeze vials by placing in an -80C freezer overnight and then into liquid nitrogen for long-term storage.
Seeding (For Experimentation) For Myoblasts*: 1. Dilute cells with Growth medium to 8 X 10 4 / ml and seed at a density of: a. 24 well plate - add 0.5 ml/well b. 6 well plate - add 2 ml/well c. 10 cm Dish - add 10 ml/dish d. 15 cm Dish - add 30 ml/ dish 2. First medium change after 48 hrs; then repeat media change every 48h. N.B. *Grow myoblasts until confluence before experimentation. Use within 12 hours after confluence. For Myotubes*: 1. Dilute cells with Differentiation medium to 4 X 10 4 / ml and seed at a density of: a. 24 well plate - add 0.5 ml/well b. 6 well plate - add 2 ml/well c. 10 cm Dish - add 10 ml/dish e. 15 cm Dish - add 30 ml/ dish 2. First medium change after 48 hrs; then repeat media change every 48h.
To visualize cells for fusion (Cells in 24-well plate): 1. Remove medium and rinse with saline 2. Completely remove saline and let cells dry for 15 min. 3. Add trypan blue solution (2-fold dilution of the original solution), leave for 3 min., remove trypan blue 4. Rinse once with saline and remove. 5. Nuclei and cell contours stain 6. Count presence of fused cells by counting nuclei, and number of non-fused cells. N.B. Fusion is considered a cell formed from 2 or more myoblasts. Another assay of fusion: Giemsa staining (stains the nuclei a deep brown colour and helps one see multinucleated cells very easily. The ready-to-use stain is sold by Sigma, catalog number GS-500) Assay of differentiation: Increased creatine kinase activity: Possonneau, JV and Lowry OH. A collection of enzyme assays. In Enzymatic Analysis. Totowa, NJ, USA: Humana 1993, Chapter 7 pp246-248. References: 1. Kishi, K., N. Muromoto, Y. Nakaya, I. Miyata, A. Hagi, H. Hayashi, and Y. Ebina. Bradykinin directly triggers GLUT4 translocation via an insulin- independent pathway. Diabetes 47: 550-558, 1998. 2. Mitsumoto, Y., E. Burdett, A. Grant, and A. Klip. Differential expression of the GLUT1 and GLUT4 glucose transporters during differentiation of L6 muscle cells. Biochem. Biophys. Res. Commun. 175: 652-9, 1991. 3. Mitsumoto, Y., and A. Klip. Development regulation of the subcellular distribution and glycosylation of GLUT1 and GLUT4 glucose transporters during myogenesis of L6 muscle cells. J. Biol. Chem. 267: 4957-4962, 1992. 4. Wang, Q., Z. Khayat, K. Kishi, Y. Ebina, and A. Klip. GLUT4 translocation by insulin in intact muscle cells: detection by a fast and quantitative assay. FEBS Lett. 427: 193-197, 1998. 5. Wang, Q., R. Somwar, P. J. Bilan, Z. Liu, J. Jin, J. R. Woodgett, and A. Klip. Protein kinase B/Akt participates in GLUT4 translocation by insulin in L6 myoblasts. Mol. Cell. Biol. 19: 4008-4018, 1999.
Helpful hints when growing L6 1-When thawing warm the vial to 37C and add to 10 ml of growth media in a 75 cm2 flask and allow to plate down for 2 hours 2-Ensure that the L6 have adhered and carefully replace the media with about 12 ml of media. Within two days the flask could be 50% confluent. I would split this flask into 5 new flasks and allow them to grow to 50 % and split all 5 another 5 flasks each (total, 25 flasks). Allow them to grow to 60-70% confluence and then freeze down 2 vials from each flask according to the protocol. 3-Care has to be taken that the myoblasts are never allowed to reach confluence when you are expanding cells for freeze-down or seeding for experimentation because this will affect their ability to differentiate into myotubes in subsequent passages. 60-70 % confluence is a good set point at which to passage the myoblasts. You should be able to carry a single vial of L6 cell for about 12-15 passages before their ability to differentiate into myotubes begins to decline. At that time start another vial of cells. 4-When seeding myoblasts for experimentation (e.g. in preparation for transfection) seed them at about 50-60% confluence for whatever size dish or cell well you are using (or according to cell number directives that may be in our protocol e.g. 1.6 x 10 4 cells into one well of a 6-well plate is about 50-60% confluent) directly into differentiation media (containing 2% FBS). You should be able to use the myotubes 8 days after cell seeding. L6 spontaneously differentiate the differentiation medium. 5- This is another helpful suggestion. It can be difficult to ensure that you have a lot of cells for seeding experiments when we suggest that one should passage when they are around 60-70% confluent. To increase your cell density without having to carry a lot of half empty flasks it is possible seed a flask into 10%FBS-containing media and allow it to go to 90% confluence. Then from this flask you can seed dishes or plates for experimentation at the cell density that we suggest above. Don't use the cells from this high-density flask to continue carrying the line. However, the cells seeded for experimentation will fuse well. Thus, keep a flask at 60-70% confluence throughout the run of a current cell cohort (12-15 passages) that can be used to seed high-density flasks or plates and dishes for experimentation.