HELA S3-FlpIn cell line growth
Aim: This protocol is meant to successfully generate numerous stable HeLa S3-FlpIn cell lines using the HeLa S3-FlpIn clone 11 (medium expression level) but should also work for the high expressing (clone 6) and low expressing (clone 13) clones. The main objective is to make HeLa S3 cells express EXOSC10-3xFLAG.
Materials: - Dulbecco’s Modiefied Eagle Medium (DMEM) - Fetal Calf Serum (FCS) - Lipofectamine LTX - Trypsin - Zeocin - Blasticidin - Hygromicin
Methods: HeLa S3 cells can be grown in suspension culture in RPMI 1640 medium or adherent in DMEM medium. For the selection of stable clones HeLa S3-FlpIn cells are grown as adherent cultures in flasks/dishes in DMEM. HeLa S3 cells kept in adherent culture grow as “spheres” but attach well to the dish.
- Thaw HeLa S3 cells and seed into a flask in DMEM + Pen/Strep/Glutamine + 10% FCS
- The next day: exchange the medium against DMEM + Pen/Strep/Glutamine + 10% FCS + 200 μg/ml Zeocin.
- Split cells 1:5 once or twice (keep in DMEM + Pen/Strep/Glutamine + 10% FCS + 200 μg/ml Zeocin).
- The day before the transfection: harvest cells by trypsinization, count and seed in 35mm dishes or 6-well plates at 1x106 cells per dish/well in a volume of 4 ml of DMEM + Pen/Strep/Glutamine + 10% FCS (Use medium without Zeocin. Zeocin is a DNA intercalator and even traces of Zeocin during the transfection will drastically reduce transfection efficiencies!)
- The next day: transfection of cells using Lipofectamine LTX (Life Technologies/Invitrogen) according to the manufacturer’s instructions. The following conditions have worked well: 2.5 μg DNA total/8.76 μl Lipofectamine.
- Dilute 0.25 μg of your pcDNA5/FRT/TO expression construct and 2.25 μg of pOG44 in 1 ml of Optimem I + Glutamax without serum! (This way the ratio of the Flp recombinase expression vector vs. the pcDNA5/FRT/TO expression construct is 9:1). Include a separate transfection of a pcDNA5/FRT/TO-GFP plasmid (with pOG44) as control for the transfection efficiency.
- Add 8.76 μl of Lipofectamine LTX and mix gently by pipetting up and down several times, then incubate the mixture at RT for 25 minutes.
- While the DNA/Lipofectamine complexes are forming: remove medium from the cells, wash cells once with DMEM + 5% FCS + Glutamine, and then replace wash medium with DMEM + 5% FCS + Glutamine (Do not include any antibiotics (Pent/Strep and Zeocin) in the wash and transfection medium at this point!)
- After the formation of the transfection complexes, add the 1 mL of DNA/Lipofectamine/Optimem mix dropwise to the cells and mix by gently moving the dish/plate back and forth several times
- Incubate at 37ºC in CO2 incubator for 24h
- The next day: exchange the transfection medium against 4 ml of DMEM + Pen/Strep/Glutamine + 10% FCS. At this point, successful transfection of the GFP-expressing cells can be checked using a fluorescence microscope. The expected transfection efficiency is around 20% for HeLa S3-FlpIn (as judged by the number of green cells in the pcDNA5/FRT/TO-GFP transfection control)
- The next day or when the cells in the dish/well are about to become confluent: trypsinize the cells and seed all cells in 20 mL DMEM + Pen/Strep/Glutamine + 10% FCS in a 15cm TC-dish
- The next day: start selection of FlpIn clones by exchanging the medium against 20 mL DMEM + Pen/Strep/Glutamine + 10% FCS + 400 μg/mL Hygromycin B
- Select stably integrated clones until individual colonies become clearly visible by eye. This will take 3-4 weeks (maybe longer) depending on the growth rate of the cells
- For polyclonal selection: trypsinize the cells to harvest and pool all colonies and expand in DMEM + Pen/Strep/Glutamine + 10% FCS + 400 μg/mL Hygromycin B. Once expanded, freeze the desired number of cells in suitable aliquots.
- For selection of clones: trypsinize several individual colonies using cloning disks or cloning rings and transfer the harvested cells from each colony into a separate well of a 24-well plate. Expand individual clones in DMEM + Pen/Strep/Glutamine + 10% FCS + 400 μg/ml Hygromycin B. When confluent first split into a 6-well and then into increasingly larger dishes/flasks. Once expanded, freeze the desired number of cells for each clone in suitable aliquots.
Both polyclonal and monoclonal populations need to be checked for the expression of the inserted gene and loss of β-galactosidase expression. Monoclonal cell lines should also be checked for loss of Zeocin resistance.
Results: For antibiotic selection I thaw HeLa cells and split them in 4 T75 flask with the following 4 different conditions: - NO Zeocin + + 5 µg/ml Blasticidin - 50 µg Zeocin + 5 µg/ml Blasticidin - 100 µg Zeocin + 5 µg/ml Blasticidin - 200 µg Zeocin + 5 µg/ml Blasticidin At the end I trashed 200 µg Zeocin as cells were not growing and I made stock of 50 and 100 µg Zeocin before quarantine.