I need this to do glucose uptake assay using 3T3-L1 cells. I checked 2 kit protocols; one by cosmo-bio and other by sigma. Both companies have suggested significantly different buffer composition (pH IS 7.4) for both.
Glucose uptake kits cited are by Cosmo-bio and other Sigma
KRPH buffer composition
HEPES - 20 mM
KH₂PO₄ - 5 mM
MgSO₄ - 1 mM
CaCl₂ -1 mM
NaCl- 136 mM,
KCl - 4.7 mM,
pH 7.4
Good luck!
20mM hepes,136mM NaCl, 4.7mM KCl, 1mM NaH2PO4,1mM MgSO4, and imM CaCl2,pH 7.4 (KRPH buffer)...Riya...any buffer without calcium and magnesium can be used for flow cytomety...I use HBSS without calcium and magnesium..but it has glucose. You can use HBSS without glucose or PBS with 1% FBS for your specific glucose uptake assay
0.1 M Krebs-Ringer-Phosphate( KRP) buffer (pH:- 7.2-7.4)
Reagents required for making KRP buffer:
• NaCl 1.5 M
87.66 g NaCl in 1 Litre deionized water.
• Phosphate buffer pH 7.4:
A) 0.1 M NaH2PO4. 2H2O (MW 156) - 15.6 g in 1 Litre.
B) 0.1 M Na2HPO4. 2H2O (MW 178) - 17.8 g in 1 Litre.
Add 19 ml of A to 81 ml of B, adjust pH to 7.4.
• KCl 0.15 M
5.6 g KCl (MW 74.56) in 500 ml distilled water.
• MgSO4. 0.15 M
3.7 g MgSO4.7H2O (MW 246.48) in 100 ml distilled water.
• CaCl2 0.11 M
1.62 g CaCl2.2H2O (MW 147.02) in 100 ml distilled water.
1. Make up 100 ml of 1.5 M NaCl to 1 litre.
2. Add: 120ml of phosphate buffer pH 7.4, 40 ml of 0.15 M KCl, 10 ml of 0.15 M MgSO4, 10 ml of 0.11 M CaCl2 – stir carefully to avoid precipitation.
3. Adjust pH with NaOH (1 M)
Krebs-Ringer-Phosphate( KRP) buffer :
a.0.1 M NaH2PO4.2H2O (M.Wt:156) 15.6 g in 1Litre (PH:7.2-7.4)
b.0.1M Na2HPO4.2H2O (M.Wt:178) 17.8 g in 1 Litre
Add 19 ml of A to 81ml of B , adjust PH to 7.4
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