KSOM media
Modified from:
G. T.Erbach . J.A. Lawitts, V. E. Papaioannou, J.D. Biggers
Differential Growth of the Mouse Preimplantation Embryo in Chemically
Defined Media, Biology of Reproduction, 50, 1027-1033 (1994)
J.A.Lawitts, J.D.Biggers
Cultute of Preimplantation Embryos
(The Methods in Enzymology, v. 225, 1993 , 153-165)
| Component | Concentration (mM) | g/L |
| NaCl | 95.0 | 5.55 |
| KCl | 2.50 | 0.186 |
| KH2PO4 | 0.35 | 0.0476 |
| MgSO4 | 0.20 | 0.0493 |
| Na.lactate | 10.00 | 1.87 |
| glucose | 0.20 | 0.036 |
| Na.pyruvate | 0.20 | 0.022 |
| NaHCO3 | 25.0 | 2.10 |
| CaCl2 (dihydrous) | 1.71 | 0.251 |
| glutamine | 1.0 | 0.146 |
| EDTA (tetrasodium salt) | 0.01 | 0.0038 |
| BSA | 1.0 mg/mL | |
| penicillin-G | 100U/mL (60µg/mL) | |
| streptomycin.sulfate | 50µg/mL |
Six stock solutions are prepared:
Stock A' (10X) (100mL)
| Component | Conc. MW | mass |
| NaCl | 950mM 58.44g/mol | 5.55g |
| KCl | 25mM 74.55g/mol | 0.186g |
| KH2PO4 | 3.5mM 136.09g/mol | 0.0476g |
| MgSO4.7H2O | 2.0mM 246.47g/mol | 0.0493g |
| Na.lactate | 100mM 112.10g/mol | 1.87g of 60% syrup |
| D (+) glucose | 2.0mM 180.20g/mol | 0.036g |
| penicillin-G | 600µg/mL | 0.06g |
| streptomycin.sulfate | 500µg/mL | 0.05g |
Freeze in 10mL aliquots.
Stock B (10X) (100mL)
| Component | Conc. MW | mass |
| NaHCO3 | 250mM 84.01g/mol | 2.10g |
| Phenol Red | 0.001 g or 1 ml of 1% solution |
Stock C' (100X) (10mL)
| Component | Conc. MW | mass |
| Na.pyruvate | 20mM 110.0g/mol | 0.022g |
Stock D (100X) (10mL)
| Component | Conc. MW | mass |
| CaCl2 2H2O | 17.1mM 147.02g/mol | 0.25g |
Stock F (10,000X) (10mL)
| Component | Conc. MW | mass |
| Na2EDTA.2H2O | 100mM 372.24g/mol | 0.372g |
| Component Conc. | |
| glutamine (Gibco# 320-1140 AG) | 200mM (in solution form) |
To make 100mL of KSOM media:
| Stock Solution | Volume |
| 10X Stock A' | 10mL |
| 10X Stock B | 10mL |
| 100X Stock C' | 1mL |
| 100X Stock D | 1mL |
| 10,000X Stock F | 10 µL |
| 200X Stock G | 0.5mL |
| solid BSA | 0.10g |
| ddH2O | up to approx.90mL |
Rinse all pipettes, tubes thoroughly into final flask. Add BSA (Bovine Serum Albumin) - Sigma A4378 or A3311- embryo tested. Allow to dissolve and gently swirl the medium without excessive frothing using magnetic stirrer.Gas with 5% CO2 in air to adjust pH to 7.4, alternatively, incubate the solution at 37oC/5% CO2 for several hours (or overnight) with loose cap. Bring the volume up to 100mL, filter through 0.2µm and aliquote into tubes. Store at +4 C for no longer than 2 weeks. To test the medium and culture conditions, collect 1 cell stage embryos(0.5 dpc) and culture them through blastocyst stage (around 100% reach this stage).