(bicarbonate-free, phenol-red free)
10X HBSS base solution:
- calcium chloride anhydrous (CaCl2 MW 111): 1400 mg/L = 12.6 mM
- OR calcium chloride dihydrate (CaCl2-2H2O; MW 147): 1854 mg/L = 12.6 mM
- magnesium chloride hexahydrate (MgCl2-6H2O; MW 203): 1000 mg/L = 4.9 mM
- magnesium sulfate heptahydrate (MgSO4-7H2O; MW 246): 1000 mg/L = 4.1 mM
- potassium chloride (KCl; MW 75): 4000 mg/L = 53.3 mM
- potassium phosphate monobasic (KH2PO4; MW 136): 600 mg/L = 4.4 mM
- sodium chloride (NaCl; MW 58): 80000 mg/L = 1379.3 mM
- sodium phosphate dibasic heptahydrate (NaHPO4-7H2O; MW 268): 900 mg/L = 3.4 mM
- D-glucose (dextrose; MW 180): 10000 mg/L = 55.6 mM
To make 1x solution, dilute 100 ml of the 10x stock into ~750 ml millipore H2O with stirring. Add 20 ml 1M HEPES (free acid; pH adjusted to 7.4 with NaOH) solution for a final concentration of 20 mM.
Optional additions (depending on experimental protocol) include:
- 1X MEM vitamins (purchased stock solutions are usually 100x); not recommended when using fura-2 due to background autofluorescence
- 1X MEM amino acids (purchased stocks are usually 50x); absolutely necessary for DAF-FM experiments where arginine is needed for NO production
- 1X MEM non-essential amino acids (purchased stocks are usually 100x)
- Sodium bicarbonate (NaHCO3) to a final concentration of 25 mM if performing experiments in the presence of 5% CO2 (solution must be thoroughly gassed with 5% and checked for pH). A 10x stock of sodium bicarbonate (250 mM) can be made in H2O.
- 1x cell culture pen/strep antibiotic mix