2-Mercaptoethanol (Sigma; M-7522)
Concentration of stock solution is 14.3 M (pure liquid), for Preparation working solution of 2-mercaptoethanol, dissolve 70 µl of 2-mercaptoethanol stock solution (14.3 M) in 10 ml of PBS; sterilize by filtration and store in a dark, cool place.
2-mercaptoethanol is a reducing agent that can break down many toxic metabolites produced by cells in cultures, thus improving the environment surrounding the cells. Mouse cells are indeed very sensitive to oxidative metabolites; therefore 2-mercaptoethanol is principally used in culturing these cells.
Bisbenzimide H 33258, (Calbiochem, 382061, 100 mg)
Dulbecco´s phosphate-buffered saline without Mg2+ and Ca2+ (D-PBS-) (Sigma; D5652). Do not heat, sterilize immediately by filtration.
DMSO (Sigma, Hybrimax, sterile–filtered; D2650)
EGTA powder (Sigma; E-4378)
EDTA has a high affinity for magnesium and a lower affinity for calcium, whereas EGTA has a high affinity for calcium and a lower affinity for magnesium. Working solution of EGTA was prepared at concentration of 2mM.
Embryonic stem cell qualified Fetal calf serum (ES-FCS) (Gibco; 16141-079)
FCS may contain undefined factors that can promote differentiation of ES cells, thus each lot must be screened prior its use in order to find the best lot quality.
Fetal bovine serum (FBS) (Invitrogen, 10270-106)
Store frozen at -20ºC. Thaw in a 37ºC water bath before adding to the culture medium. Once thawed, FBS will remain stable at 4ºC for 3–4 weeks.
Avoid repeated refreezing FBS which has been thawed.
Gelatin (Sigma; G2500)
Gelatin is a heterogeneous mixture of water-soluble proteins with high average molecular weight that are present in collagen. The change on a gelatin molecule and its isoelectric point are primarily due to the carboxyl amino and guanidine groups on the side chains of it.
L-glutamine solution (Gibco; 25030-024)
200 mM sterile solution, cell culture tested.
Leukemia inhibitory factor (LIF) (ESGROTM; 13275)
This cytokine is often used to assist with maintaining the pluripotency of mESCs.
Mitomycin C powder (Sigma; M0503)
Prepare fresh before use. Notice that the amount of Mit-C in a culture medium (at 38ºC) that contains antibiotics and serum, decreases over time.
Non-essential amino acid (Gibco; 11140-035). Store aliquots in fridge
Thymidine powder (Sigma; T1895)
Trypsin-EDTA (1x) in HBSS without Mg2+ and Ca2+ (Gibco; 25300-054)
The optimal activity of trypsin is in a partially alkaline (pH 7.8-8.7) environment with no Mg2+ and Ca2+ ions.
BD Matrigel™ (BD Biosciences; cat. #354277)
Since Matrigel™ matrix forms a gel above 10°C; it should be kept at a low temperature. Therefore, all equipment and reagents (tips, Matrigel™ matrix solution, etc.) should be chilled on ice prior to use. For the preparation of Matrigel™, add 350 µl from the stock solution to 6 ml of cool medium. Thaw Matrigel™ at 4ºC.
Cytochalasin-B (Sigma; C6762)
Dissolve 5 mg solid Cytochalasin-B in 1 ml DMSO to give a Cyt-B concentration of 5000 µg/ml as follows (1000x solution):
i. Remove the cyt-B vial from -20ºC and place at room temperature. Do not remove the seal.
ii. Sterilize the top of the rubber seal with 70% ethanol and allow the ethanol to evaporate.
iii. Vent the vial’s seal aseptically. With a 2.5 ml sterile syringe, inject 1 ml DMSO through the seal.
iv. Mix contents gently. Cytochalasin-B dissolves readily in DMSO.
v. Mix and dispense 50 µl of 1000x stock solution aliquots into sterile, 0.5 ml polystyrene tubes. Date and label tubes with “cyt-B”.
vi. Store at -20ºC for up to 12 months. The vials of powder are guaranteed by Sigma for 2 years if stored at -20ºC.
vii. For preparation of 100x and 10x cyt-B, add 450 µL and 4950 µL DMEM, respectively.
Cytochalasin-B is toxic and it can be a possible teratogen agent. It must always be purchased in sealed vials. The preparation of this reagent must be carried out in a cytoguard cabinet.
DMEM: The culture medium needed for feeder layer growth.
- DMEM (Gibco; 12800 -116) 13.5 g/l
- NaHCO3 (Sigma; S-5761) 3.7 g/l
- Penicillin/Streptomycin (Gibco; 15070-063) 10 ml/L
- HCl (1 N) 3 ml/L
- 2-mercaptoethanol (Sigma; M-7522) 7 µL
Add these components to 837 ml of cell culture grade de-ionized water filter and store in dark, cool conditions. Adjust the pH to 0.2-0.3 units below the desired working pH (7.0-7.4) by adding 1N NaOH or 1N HCl with stirring. The pH of bicarbonate buffered solutions usually raises 0.1–0.2 units during filtration.
- Knock-out™ DMEM (Gibco; 10829-018) 500 ml
- Penicillin/streptomycin (Gibco; 15070-063) 5 ml
- Non-essential amino acid (Gibco; 11140-035) 5 ml
- 2-mercaptoethanol (Sigma; M-7522) 0.5 ml
- ES-FCS (Gibco; 16141-079) 75 ml
Aliquot and store at 4ºC in a dark place. For 50 ml KO-DMEM, 500 µl of L-glutamine and 50 µl LIF  are needed.
Periodic discontinuation of antibiotic use is recommended for several reasons. First, questionable cell culture techniques can be masked; antibiotic-resistant organisms may arise, and finally, cryptic microbial contamination may be present and masked by antibiotic use.
Gelatin treatment of flasks
i. Add 3-4 ml of 0.1% sterile gelatin to cover the bottom of the T-25 flask.
ii. Let the gelatin sit for 1 hour at 37ºC. Sitting longer is also acceptable.
iii. Remove excess fluid from the coated surface and allow to air dry under a laminar flow hood for approximately 1/2 hour (alternatively can allow to air dry overnight).
iv. Rinse with sterile tissue culture grade water or a balanced salt solution before adding the media and/or cells (optimal condition for attachment must be determined for each cell line and application).
Hoechst 33258 stock solution Preparation:
Stock solutions of most of the dyes are prepared in PBS at concentrations of 1.0 mg/ml. However, DAPI and the Hoechst dyes should be prepared in distilled water since at relatively high concentrations these dyes tend to precipitate in PBS. Stock solutions are refrigerated in dark-colored containers or wrapped in foil 7.
This material is harmful if inhaled, by contact with skin and if swallowed.
The Bisbenzimide Hoechst dyes are designated by numbers 33258, 33342 and 34580. As with H33258, Hoechst stain H33342 is used for staining DNA. It binds to AT-rich sequences (in the minor groove) without intercalation 8. H 33342 exhibits 10-fold greater cell permeability than H 33258.
DAPI and H 33258 only stain vital cells, while H 33342 stains vital and dead cell types 9, with blue emission when examine with fluorescent microscopy. H 33258 gives a more stable reaction compared with Hoechst 33342 10. The dye is excited at 365 nm and the filter (emission) is 418 nm 8.
Mitomycin-C treatment of mouse embryonic fibroblast cells (mEF)
i. Aspirate media from day 14 mEF that are confluent in a T-75 flask.
ii. Add 200 µl Mit-C to 10 ml of culture medium and incubate for 90 minutes at 37ºC.
iii. Aspirate off the media and wash three times with 5 ml of PBS- to remove any trace of Mit-C.
iv. Trypsinize and resuspend the cells in growth medium and add 2.5x106 cells to each pre-gelatinized T-25 flask that contains growth medium adjusted to the appropriate pH. Rotate and distribute the cell suspension evenly on the surface of the flask. Cell attachment of fibroblasts cells usually takes place within 1-2 hours.
Cells that are being passaged are unable to adjust to the pH of the medium for an amount of time prior to attachment and re-initiation of metabolic activity. The passage time is a particularly critical period that can be achieved by prewarming the flask that contains growth medium in a CO2 incubator or aseptically gassing each flask with 5% CO2.
Mitomycin-C should be stored in the dark since it is readily decomposed by light.
i. Appropriate protection must be done while working with mitomycin-c. Wearing suitable protection is recommended.
Slides are mounted in PBS/glycerol (1:1).
First, check components for auto fluorescence. Residual auto fluorescent mounting medium can be removed by the offset of imaging software.
Once aqueous mounting media is made, it should be stored in small (10-30 ml) screw-capped bottles or vials made of transparent glass or plastic.
Preparation of thymidine
ii. Dissolve 0.1816g of thymidine powder in 10 ml PBS to give a 75 mM thymidine solution (stock solution).
iii. Add 100 µl of stock solution to 3 ml of culture medium (final thymidine concentration is 2.5 mM).