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Red Blood Cell Lysis Buffer (ACK Lysis Buffer)

Instruction manual

First Edition (Revised on April, 2016)

[PRODUCT INFORMATION]

The Red Blood Cell Lysis Buffer could be used for lysis and removal of red blood cells from blood or tissue samples while leaving white blood cells intact. 


[REAGENT]

REAGENT

Quantity

Red Blood Cell Lysis Buffer

20ml/50ml/100ml


[STORAGE AND PERIOD OF VALIDITY]

Store at 4°C for one year.


[ASSAY PROCEDURES]

A. For tissue/cell samples:

1. Fresh tissues are digested with collagenase or pancreatin, dispersed into cells suspension by appropriate methods, centrifuge and discard the supernatant.

2. For every 1 ml of cell pellet, add 3-5 ml of Red Blood Cell Lysis Buffer. Mix gently by pipetting, and lyse for 1-2 minutes. Please operate at room temperature or 4°C.

3. Centrifuge at 400-500g for 5 minutes at 4°C and discard the red supernatant.

4. Repeat steps 2 and 3 if the erythrocyte lysis is incomplete. In general, a very small amount of erythrocyt will not affect following tests.

5. Wash 1-2 times: add appropriate volume of PBS, HBSS, 0.9% NaCl or serum-free media, resuspend the precipitates, centrifuge at 400-500g for 2-3 minutes at 4°C, and discard the supernatant. The volume of wash solution should usually be at least 5 times of the volume of cell precipitates.

6. Resuspend the cell precipitates with appropriate reagent according to the experiment.

 

B. Quick operation for tissue/cell samples without washing:

1. Fresh tissues are digested with collagenase or pancreatin, dispersed into cells suspension by appropriate methods, centrifuge and discard the supernatant.

2. For every 0.2 ml of cell pellet, add 1 ml of Red Blood Cell Lysis Buffer. Mix gently by pipetting, and lyse for 1-2 minutes. Please operate at room temperature or 4°C.

3. Add 15-20ml of PBS, HBSS, 0.9% NaCl or serum-free media, mix well.

4. Centrifuge at 400-500g for 5 minutes at 4°C and discard the red supernatant.

5. Repeat steps 2 and 3 if the erythrocyte lysis is incomplete. In general, a very small amount of erythrocyte will not affect following tests.

6. Resuspend the cell precipitates with appropriate reagent according to the experiment.

Note: The quick operation needs large volume of centrifuge tubes, this operation reduces the contrifuge step with slightly lower washing efficiency.

 

C. For blood samples:

1. Centrifuge fresh anticoagulated blood at 400-500g for 5 minutes and discard the supernatant.

2. For every 1 ml of cell pellet, add 6-10 ml of Red Blood Cell Lysis Buffer. Mix gently by pipetting, and lyse for 1-2 minutes. Please operate at room temperature or 4°C. 

Note: For rat/mouse blood, lyse for 1-2 minutes is sufficient. For human peripheral blood, prolong the lysis time to 4-5 minutes.

3. Centrifuge at 400-500g for 5 minutes at 4°C and discard the red supernatant.

4. Repeat steps 2 and 3 if the erythrocyte lysis is incomplete. In general, a very small amount of erythrocyte will not affect following tests.

5. Wash 1-2 times: add appropriate volume of PBS, HBSS, 0.9% NaCl or serum-free media, resuspend the precipitates, centrifuge at 400-500g for 2-3 minutes at 4°C, and discard the supernatant. The volume of wash solution should usually be at least 5 times more than the volume of cell precipitates..

6. Resuspend the cell precipitates with appropriate reagent according to the experiment.

Note: For small amounts of blood samples, the first step is not necessary, you can directly add 10 ml of Red Cell Lysis Buffer for every 1 ml of cell pellet in step 2 and lyse for 4-5 minutes at room temperature or 4°C. For rat/mouse blood, lyse for 4-5 minutes is sufficient. For human peripheral blood, prolong the lysis time to 10 minutes, but do not exceed 15 minutes. The following steps are the same.

 

D. Quick operation for blood samples without washing:

1. For every 1 ml of fresh anticoagulated blood, add 10 ml of Red Blood Cell Lysis Buffer. Mix gently by pipetting, and lyse for 4-5min. Please operate at room temperature or 4°C.

Note: For rat/mouse blood, lyse for 4-5 minutes is sufficient. For human peripheral blood, prolong the lysis time to 10 minutes, but do not exceed 15 minutes.

2. Add 20-30ml of PBS, HBSS, 0.9% NaCl or serum-free media, mix well.

3. Centrifuge at 400-500g for 5 minutes at 4°C and discard the red supernatant..

4. Repeat steps 2 and 3 if the erythrocyte lysis is incomplete. In general, a very small amount of erythrocyte will not affect following tests.

5. Resuspend the cell precipitates with appropriate reagent according to the experiment.

Note:The quick operation needs large volume of centrifuge tubes, this operation reduces the contrifuge step with slightly lower washing efficiency.


[IMPORTANT NOTES]

1. Reagents should be stored according to the instructions.

2. For your safety and health, please wear a lab-gown and disposable gloves in experiments.