Modified Phosphate-Buffered Saline (PBS) Supplemented with Glucose, Sodium Pyruvate, BSA, and Antibiotics

• 1. Dissolve the PBS packaged chemicals (e.g., Sigma-Aldrich D-5773 or similar) in 800 mL of H₂O.

• 2. Dissolve and add the CaCl₂ solution.

• 3. Bring volume to 1 L.

• 4. Filter-sterilize and store for up to 1 mo at 4°C.

• 5. To 100 mL of PBS add the following:

  Reagent	Catalog no.	Amount
  d-Glucose	Sigma-Aldrich G6152	100 mg
  Sodium pyruvate	Sigma-Aldrich P4562	3.6 mg
  Penicillin G	Sigma-Aldrich P4687	7.5 mg
  Streptomycin sulfate	Sigma-Aldrich S1277	5.0 mg
  BSA (embryo tested)	Equitech-Bio BAC62-0050 (for example)	300 mg

• 6. Allow BSA to dissolve, do not shake.

• 7. Filter-sterilize and measure osmolarity (should be 280 mOsm ± 5%).

• 8. Keep refrigerated and use within 2 wk.

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