RBC Lysing Solutions
RBC Lysing Solutions and Cell Lysing Procedure
Ammonium chloride lyse (10X concentration)
NH4Cl (ammonium chloride) 8.02gm
NaHCO3 (sodium bicarbonate) 0.84gm
EDTA (disodium) 0.37gm
QS to 100ml with Millipore water. Store at 4 oC for six months.
Working solution
Dilute 10ml 10X concentrate with 90 ml Millipore water. Refrigerate until use.
Procedure
1.Obtain a whole blood specimen in a heparin tube.
2.Aliquot 1ml blood into 15ml conical centrifuge tube.
3.Fill tube to capacity with fresh cold lysing solution.
4.Invert or rock for ~10 minutes at room temperature until liquid is clear red.
5.Spin at 4C for 10 minutes at 250 x g.
6.Decant supernatant and allow tubes to drain briefly.
7.Resuspend cell by raking gently across a tube rack.
8.Wash cells and combine multiple tubes with 10ml cold PBS/2% FCS.
9.Spin, decant, and resuspend as above (steps 5-7).
10. Count cells and adjust cell concentration to ~2-4x 106/ml.
ERYTHROCYTE LYSING SOLUTION-10X STOCK SOLUTION (peripheral blood)Procedure
1. Mix 14 ml 1x working solution (lysing buffer) to 1 ml Blood.
2. Incubate for 3 - 5 minutes.
3. Centrifuge at 300 x g for 5 minutes at room temperature.
4. Aspirate supernatant and resuspend pellet in 5 ml of COLD Phosphate Buffered Saline(PBS).
5. Centrifuge at 300 x g for 5 minutes at 2 - 8°C.
6. Aspirate supernatant and resuspend pellet in 1 ml of COLD PBS with protein carrier
10X Stock Lysing Buffer
NH4Cl 89.9 g
KHCO3 10.0 g
Tetrasodium EDTA 370.0 mg
Milli Q Water 1.0 Liter
Adjust pH to 7.3. Filter Sterilized. Store at 4o
C in full, tightly closed 50 ml tubes.
1X Working Solution (lysing buffer)
Add 5 ml 10X Stock Lysing Buffer to 45 ml H2O. Mix well. Store at room temperature for up to one week
1X ACK (Ammonium Chloride Potassium)
155mM NH4Cl 8.29g
10mM KHCO3 1.0g
0.1mM EDTA 0.2 ml (500mM)
RBC lysis buffer 10x
1. Prepare 8,26 g of NH4Cl
2. Add 1,19 g of NaHCO3
3. Add 200 µL of EDTA [0,5 M, pH8]
4. Add a.d until 100 mL
5. Adjust pH to 7.3
6. Filter sterile
Note: to obtain 1x solution, dilute with a.d sterile
Erythrocyte Lysing Solution (no preservative) 10X Stock Solution
Dissolve in 1 liter of H2O:
89.9 g NH4Cl
10.0 g KHCO3
370.0 mg tetrasodium EDTA
Adjust pH to 7.3. Store at 4°C in full, tightly closed 50 ml tubes.
1X Working Solution
Add 5 ml 10X Lysing Stock Solution to 45 ml H2O. Mix well. Store at room temperature for up to one week.
Procedure
Mix 14 ml Lysing Buffer to 1 ml Blood.
Incubate for 3 - 5 minutes.
Centrifuge at 300 x g for 5 minutes at room temperature.
Aspirate supernatant and resuspend pellet in 5 ml of COLD Phosphate Buffered Saline (PBS).
Centrifuge at 300 x g for 5 minutes at 2 - 8°C.
Aspirate supernatant and resuspend pellet in 1 ml of COLD PBS with protein carrier.