Preparation of Culture Media for Fertilized One Celled Eggs

Two types of media are required for the in vitro manipulations of the eggs (12).

1. M16: for maintaining the eggs in microdrop cultures in a 37°C incubator gassed with 5% CO2. M16 is buffered with bicarbonate alone and unsuitable for maintaining the eggs outside of the incubator, since the eggs are very susceptible to pH changes.

2. M2: essentially similar to M16, except that the bicarbonate is partially replaced by HEPES buffer to facilitate the survival of the eggs outside the CO2 incubator. Eggs should not be in M2 longer than 30 min.

Both M2 and M16 contain bovine serum albumin (BSA), which reduces the stickiness of the eggs and absorbs low-level poisons. Some batches of BSA are toxic to the eggs, so it is crucial that each stock is tested for its ability to sustain mouse embryo development through to the blastocyst stage (e.g., BSA; Sigma cat. no. 4161: Ho et al. [unpublished observations] in Waller et al. [8])

Media preparation:

1. Use Sigma tissue-grade chemicals throughout.

2. Make up all stocks using sterile disposable plastic containers and pipets. Washed glass items can be contaminated with detergents that are toxic to the eggs. Filter all concentrated stocks through 0.45-|jm Millipore filters into sterile plastic tubes. Store frozen at 20°C.

3. Make up the M2 and M16 culture media as follows: a. Concentrated stocks:

i. 100X A: Weigh out the following reagents in a 50-mL sterile tube: 2.767 g NaCl (Sigma S5886), 0.178 g KCl (Sigma P5405), 0.081 g KH2PO4 (Sigma P5655), 0.1465 g MgSO4 7H2O (Sigma M2643), 0.5 g glucose (Sigma G6138), 0.03 g penicillin (Sigma P3032), 0.025 g streptomycin (Sigma S9137). Weigh out 1.305 g sodium lactate (Sigma L4263) into a microcentrifuge tube and add this to the 50-mL tube. Rinse the microcentrifuge tube with double-distilled water and use the rinsings to make 10X A to 50 mL final volume ii. 10X B: Dissolve 1.0505 g NaHCO3 (Life Sciences, cat. no. 895-1810 1 N) and 0.005 g phenol red (Sigm P5530) in water and make up to 50 mL final volume iii. 100X C: 0.18 g Na pyruvate (Sigma P5280) in 50 mL water iv. 100X D: 1.26 g CaCl22H2O (Sigma C7902) in 50 mL water v. 100X E: 2.979 g HEPES (Sigma H9136), 0.005 g phenol red (Sigma P5530) into a sterile 50-mL tube and dissolve in 25-mL double-distilled water. Adjust to pH 7.4 with 0.2 M NaOH, then make up to 50 mL final volume.

b. Preparation of M2 nad M16 media from concentrated stock: To prepare M2 and M16, mix the stock solutions in the appropriate volumes as detailed below. Measure the double-distilled water in a sterile 50 plastic tube. Aliquot the concentrated stocks into the water, then carefully rinse the pipet by sucking the liquid up and down. Add the BSA and mix gently until dissolved. Pass the mixed solutions through a 0.45-|jm Millipore filter using a large sterile 60 mL disposable syringe, aliquoting into sterile containers. Store at 4°C. Prepare fresh every 2 wk.

Stock

M2

M16

10X A

5.0 mL

5.0 mL

10X B

0.8 mL

5.0 mL

100X C

0.5 mL

0.5 mL

100X D

0.5 mL

0.5 mL

10X E

4.2 mL

Double-distilled water

39.0 mL

39.0 mL

BSA (Sigma A4161)

0.2 g

0.2 g

Total volume

50.0 mL

50.0 mL

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