(M. Fero — 1/4/95, with thanks to P. Soriano)
- Glassware: All hand washed with no soap.
- Gelatin (Sigma G-1393) Diluted to 0.1% in H2O, + 1 drop of media to colorize
- Plastic pipets, P100 tissue culture dishes and 24 well multiwell dishes.
- Trypsin (Gibco 25200-023)
- BioRad cuvettes (0.4 cm, electrode gap 50, Cat# 165.2088)
- PBS (Dilute from a 10 Stock solution, autoclaved)
- 1 pk Gibco DME powder (cat #: 12100-046)
- 2.2 gm NaHCO3
- q.s. to 1 L with millipore H2O
- pH to 7.3 with ~ 5 drops of 12M HCl
- 7.5 µL ß-ME
- 5 mL Pen/Strep (Stock = 10K u/mL Pen + 10 mg/mL Strep)
- + 10 mL 200 mM Glutamine (if older than 1 mos.)
- STO cell media: DME + 10% FBS
- ES cell media: DME + 15% FBS (lot titered for ES cell toxicity)
- 2x Freezing media:
- 30 mL DME
- 10 mL FBS
- 10 mL sterile DMSO (Sigma D 2650), final concentration of 20%.
- Cryovials and cryo-freezing container
- MMC, mitomycin C (Sigma M-0503) 0.5 mg/mL in PBS, filter sterilized, light protected, 4°C
- Glass tubing (Kimble 1.2 - 1.5 mm borosilicate glass)
- Cell lysis buffer:
- KG buffer with no BSA or gelatin
- 1% ß-ME instead of 0.5% ß-ME
- 0.5% Triton X-100
- proteinase K 0.5 mg/mL.
- LIF and Neo (or Hygro) transformed STO cells
- E.S. cells
Splitting STO cells:
- Aspirate off media.
- Rinse with 6 mL of PBS, and aspirate off.
- Add 1 mL of trypsin, 37°C x 3 min.
- Add 2 mL of media to plate, and loosen up cells with a repeated pipeting. Save 1 drop to quantitate in hemocytometer while spinning.
- Spin 5 min. Suck off supernatant and resuspend cell pellet in minimal volume by flicking. Add 1-2 mL of media.
- Plate 5 x 10^4 cells/cm2 (8.8 x 10^6 cells) onto 5 large 150 mm plates with 20 mL of media. (10^-4 mL/large square in hemocytometer)
Freezing STO cells:
- Trypsinize cells as when splitting them.
- Resuspend to a concentration of 4 million cells per mL in STO cell media.
- Add 1 volume of freezing media.
- Aliquot 1 mL into cryo vials.
- Gradually cool to -20°C in a cryo container for 2 to 4 hrs.
- Transfer to liquid N2 for storage.
Mitomicin C inactivation of STO cells:
- Gelatinize and appropriate number of 100 mm plates (approximately 60 plates total will be needed to target a single ES cell construct.) Add 5 mL of gelatin solution to each plate for 20 min. Aspirate off the gelatin and allow to air dry.
- Add 0.4 mL of MMC (0.5 mg/mL stock) in 20 mL of fresh STO media to each 150 mm plate of subconfluent STO cells
- Incubate at 37°C for 2 to 4 hours.
- Rinse off MMC with PBS twice. Trypsinize and plate STO cells onto the gelatinized plates in 8 mL of media at the same density listed for splitting cells, above (4 million/100 mm plate).
- Label plates with the day of the month.
- Feed cells every 2 weeks. They are good for 1 month.
Thawing E.S. cells:
- Gelatinize a 100 mm and two 150 mm plates by covering with gelatin solution for > 1 hr. (Keep 150 mm plates for passage of STO cells.
- Quickly thaw vial of STO cells at 37°C.
- Gradualy add thawed cells to media in a 15 mL conical tube. (While gently agitating). Spin cells to pellet, then resuspend in 1 mL of media. Count with hemocytometer. (10-4 mL/Large square) Use 5 x 104 cells/cm2, or 4 x 106 cells per 100 mm dish.
- Add cells to 100 mm dish with 8 mL of STO media.
- Incubate 37°C, 5% CO2, o.n.
- Split cells the following day.
Splitting E.S. cells:
- Warm media and thaw trypsin.
- Aspirate off media. Rinse cells 1x with PBS.
- Cover cells with 0.5 mL trypsin. Incubate at 37°C for 5 min.
- Agitate plate and incubate at 37°C for 1 additional min.
- Break up cell clumps by repeated pipetting and add 0.5 mL of E.S. media to stop enzyme.
- Count cells on hemocytometer (10-4 mL / large square) and pellet remainder of cells at 1000 rpm.
- Plate 5 million cells/P100 dish (75 cm2) in 10 mL of E.S. media.
Numbers of STO and ES cells per plate
|Type of Dish
|| Well Diameter (mm)
|| Volume (mL/well)
||# STO cells (growing) (x106)
||# STO cells (inactivated) (x106)
|| # ES cells (x106 )
|6 well dish
|12 well dish
|Mini-4 well dish
Electroportating E.S. cells:
- Linearize 100 µg of the DNA construct and inactivate the restriction enzyme. Ethanol precipitate the DNA and resuspend it at a concentration to 2.5 mg/mL according to a flourimeter. (This probably corresponds to 5 mg/mL on a spectrophotometer).
- Refeed ES cells in a.m.
- Rinse off media with PBS.
- Add 2.5 mL of trypsin, incubate at 37°C for 5 min. Agitate dish and incubate for 1 min. more.
- Inactivate trypsin with 1 mL of media. Resuspend well with a transfer pipette.
- Count cells with a hemocytometer and pellet cells at 1K rpm.
- Resuspend cells at 12.5 x106 cells/ mL in PBS. (Note: Previously listed as 1.25)
- Transfer 0.8 mL to a BioRad cuvette. Add 10 µL of (2.5 mg/mL) DNA. Electroporate at 230 V. 500 µFD which should give a time constant of 5 msec.
- Transfer cells to 10 mL of ES media and plate on a single P100 plate of STO cells (for promorless constructs) or on 6 plates (for constructs containing PGK-Neo).
- On the following day, and daily therafter feed the cells with ES media supplemented with G418 (300 µg/mL) for Neo constructs, or hygromycin at 150 µg/mL. Also supplement with gancyclovir (2.5 µM, MW=255) if an HSV-TK construct is being utilized. Treat one plate with G418 alone to test the effectiveness of the TK selection. Feed cells daily with fresh pre-warmed media with the appropriate antibiotics for at least one week or until colonies clearly emerge (~10 days).
Note: If you are using a fresh batch of antibiotics or a new selection vector it may help to emperically determine the ideal drug doses. Conduct a series of 3-fold dilutions bracketing the usual drug concentration for a total of 5 doses (versus no drug). Electroporate 106 ES cells with an empty selection vector and plate them into a 6 well dish containing inactivated STO cells. Compare this to an equivalent number of ES cells electroporated with no DNA. Feed the cells daily, quantify colony formation at 10 days and observe for signs of differentiation of the colonies. Typically 1/1000 cells will stably but integrate the DNA (or about 160 colonies per well). If double drug selection is being performed (e.g. Neo + TK genes) the entire titration should be repeated using a constant G418 dose and variable gancyclovir doses using a vector known to target a specific gene. Ganciclovir typically induces a 10-fold reduction in colony formation with a targeting vector (and an equivalent enrichment of proper integration events). The efficiency of a targeting vector is exponentially related to the total length of its homology regions.
Picking E.S. cell clones for PCR screening:
- Prewarm media and PBS.
- Constuct a mouth pipette (From a mouth piece, rubber tubing, a Drummond 0.8 µm filter, a yellow tip, 1.0 mm I.D. Tygon tubing). Pull glass capillary tubes over a Bunsen burner with a low flame. Break off tubes with a 2" stem.
- Aspirate the media from plates. Invert the plate on the microscope and dot large clones (2 mm) which are well separated. Number the clones or circle them in groups of 5 to10 if pools are to be screened.
- Cover the colonies with 10 mL of PBS. Pick off 1/5 of a colony under a dissecting microscope. (Tips: Don't suck up air bubbles, hold the mouth piece in your teeth, puff on the tubing like a cigar to draw up small quantites, open your mouth to break the vacuum and cease drawing up - it will usually stay put due to capillary action. Alternatively, use our tongue to stop the capillary flow).
- Transfer the colony fragments to eppendorf tubes.
- After 15 or 20 min of picking recover colonies with warm media and return to the incubator. Alternate plates to let the cells recover.
- Spin the tubes and remove the PBS. Resuspend cells in 10 µL of Cell Lysis buffer.
- Alternatively, if single colonies are being picked in a minimal volume they can be transfered directly into tubes containing 12 µL of Cell Lysis buffer. This way there's no need to spin the cells.
- Lyse the cells by incubating at 55°C for a total of 1 hr. Vortex and spin the tubes to reduce the condensation in the middle and again at the end of the incubation time.
- Heat denature the DNA at 95°C for 5 min.
- Use 1 µL of DNA for a 20 µL PCR reaction.
Replating E.S. Clones:
- Spot out trypsin onto a sterile dish in 7 µL aliquots and cover to minimize evaporation.
- Aspirate media from the E.S. cells and cover them with 10 mL of PBS.
- Transfer the colonies of interest using the mouth pipette and a "larger bore" pulled cappillary tube in a minimal volume of PBS to the trypsin droplet.
- Incubate at 37°C for 5-10 min. The convention is to consider this an additional "passage" when counting passage numbers.
- Meanwhile give the feeder STO cells fresh E.S. media on 24 well dishes.
- Using a pipetman and gel loading tips break up the colony with repeated aspirations, and then transfer the cells to the dish of STO cells. Agitate at 90° angles to evenly distribute the cells and incubate at 37°C.
- Feed cells daily with E.S. media.
- As the cell number increase transfer to 6 well dishes and then P100s.