HBSS

HEPES-Buffered Hank's Balanced Salt Solution (HBSS) for live cell imaging at atmospheric CO₂
(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 

The 10x base solution can be purchased (Corning 20-023-CV) or made from scratch.  Dissolve solids in ~800 ml millipore H₂O with stirring.  Calcium and magnesium salts should each be dissolved separately in a small volume (~50 ml) H₂O first before adding to the other components.   Volume to 1L and filter sterilize.  Store at room temperature.  Inspect solution before every use for (1) contamination or (2) precipitation of calcium phosphate.  If precipitation of calcium phosphate is a common problem (often caused by temperature increases, though this is more common in phosphate-buffered solutions and rare in HEPES-buffered solutions), store the solution at 4°C.

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   

Adjust the pH of the 1X solution to 7.4 and volume to 1 L.  Filter sterilize.  If solution has one or more MEM components, store at 4°C.