Editing RBC Lysing Solutions

RBC Lysing Solutions and Cell Lysing Procedure

Ammonium chloride lyse (10X concentration)<br/>NH4Cl (ammonium chloride) 8.02gm<br/>NaHCO3 (sodium bicarbonate) 0.84gm<br/>EDTA (disodium) 0.37gm<br/>QS to 100ml with Millipore water. Store at 4 oC for six months.

Working solution<br/>Dilute 10ml 10X concentrate with 90 ml Millipore water. Refrigerate until use.<br/>Procedure<br/>1.Obtain a whole blood specimen in a heparin tube.<br/>2.Aliquot 1ml blood into 15ml conical centrifuge tube.<br/>3.Fill tube to capacity with fresh cold lysing solution.<br/>4.Invert or rock for ~10 minutes at room temperature until liquid is clear red.<br/>5.Spin at 4C for 10 minutes at 250 x g.<br/>6.Decant supernatant and allow tubes to drain briefly.<br/>7.Resuspend cell by raking gently across a tube rack.<br/>8.Wash cells and combine multiple tubes with 10ml cold PBS/2% FCS.<br/>9.Spin, decant, and resuspend as above (steps 5-7).<br/>10. Count cells and adjust cell concentration to ~2-4x 106/ml.

ERYTHROCYTE LYSING SOLUTION-10X STOCK SOLUTION (peripheral blood)Procedure<br/>1. Mix 14 ml 1x working solution (lysing buffer) to 1 ml Blood.<br/>2. Incubate for 3 - 5 minutes.<br/>3. Centrifuge at 300 x g for 5 minutes at room temperature.<br/>4. Aspirate supernatant and resuspend pellet in 5 ml of COLD Phosphate Buffered Saline(PBS).<br/>5. Centrifuge at 300 x g for 5 minutes at 2 - 8°C.<br/>6. Aspirate supernatant and resuspend pellet in 1 ml of COLD PBS with protein carrier<br/>10X Stock Lysing Buffer<br/>NH4Cl 89.9 g<br/>KHCO3 10.0 g<br/>Tetrasodium EDTA 370.0 mg<br/>Milli Q Water 1.0 Liter<br/>Adjust pH to 7.3. Filter Sterilized. Store at 4o<br/>C in full, tightly closed 50 ml tubes.<br/>1X Working Solution (lysing buffer)<br/>Add 5 ml 10X Stock Lysing Buffer to 45 ml H2O. Mix well. Store at room temperature for up to one week

1X &nbsp;ACK (Ammonium Chloride Potassium)

&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; 155mM NH4Cl &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;8.29g

&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; 10mM KHCO3 &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;1.0g

&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; 0.1mM EDTA &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;0.2 ml (500mM)

RBC lysis buffer 10x<br/>1. Prepare 8,26 g of NH4Cl<br/>2. Add 1,19 g of NaHCO3<br/>3. Add 200 µL of EDTA [0,5 M, pH8]<br/>4. Add a.d until 100 mL<br/>5. Adjust pH to 7.3<br/>6. Filter sterile<br/>Note: to obtain 1x solution, dilute with a.d sterile&nbsp;

<br/>Erythrocyte Lysing Solution (no preservative) 10X Stock Solution<br/>Dissolve in 1 liter of H2O:

89.9 g NH4Cl<br/>10.0 g KHCO3<br/>370.0 mg tetrasodium EDTA<br/>Adjust pH to 7.3. Store at 4°C in full, tightly closed 50 ml tubes.<br/>1X Working Solution<br/>Add 5 ml 10X Lysing Stock Solution to 45 ml H2O. Mix well. Store at room temperature for up to one week.

Procedure<br/>Mix 14 ml Lysing Buffer to 1 ml Blood.<br/>Incubate for 3 - 5 minutes.<br/>Centrifuge at 300 x g for 5 minutes at room temperature.<br/>Aspirate supernatant and resuspend pellet in 5 ml of COLD Phosphate Buffered Saline (PBS).<br/>Centrifuge at 300 x g for 5 minutes at 2 - 8°C.<br/>Aspirate supernatant and resuspend pellet in 1 ml of COLD PBS with protein carrier.


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