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Home » Research Cores » Flow Cytometry Core Facility » Protocols and Reagents » Sorting and Staining Buffers
Sorting and Staining Buffers

Sorting Buffer
 
PBS or Hanks (Ca/Mg ++ free)

25mM Hepes pH= 7.0

0.5-2% FBS (heat inactivated)

1mM EDTA.

0.2 mm sterile filtered

Notes:

Culture Media is not good as a sort buffer because outside the confines of the incubator, the pH tends to become basic, which may be the reason for low viability and recovery. The addition of Hepes will help to stabilize the pH.

For cells that don't stick together, you can modify the sort buffer to omit the EDTA. You should use the least amount of FBS the cells need to remain happy.

The addition of EDTA will help reduce the stickiness of some cell types. The concentration of EDTA should not exceed 5mM.

Staining Buffer

0.1% BSA solution in 1X PBS filter-sterilized.  Place on ice or store at 4oC until use.  You can make up 1 L at a time and store at 4oC, as long as it is kept sterile for staining cells.

10X PBS:

1.  Sodium Phosphate, monobasic, monohydrate  2.56g 
  (NaH2 PO4 -H20 (FW 137.99))   
2.  Sodium Phosphate, dibasic, heptahydrate  22.49g 
 (Na2 HPO4 -H20 (FW 268.07))   
 OR  
 Sodium Phosphate, dibasic, anhydrous 11.94g 
  ( Na2 HPO4 (FW 141.96))   
3.  Sodium Chloride  NaCl (FW 58.44)  87.66g 
  
 Bring up to 1L in d H20 and filter sterilize if needed.