Red Blood Cell Lysis Buffer (10×)

For Tissue Cell Samples (Requiring Washing)

1. Cell Collection: Fresh tissues are digested with collagenase, trypsin, or similar enzymes and dispersed into a cell suspension, and centrifuged to discard the supernatant.

2. Cell Lysis: Dilute Red Blood Cell Lysis Buffer (10×) with deionized water to 1× concentration, add 3-5 volumes of 1× Lysis Buffer to resuspend the cells, and lyse on ice or at room temperature for 1-2 min. Centrifuge at 4°C and 500 × g for 5 min, and discard the red supernatant.

Note: If incomplete lysis of red blood cells is observed upon collecting the precipitate, repeat the above steps once. Usually, a very small amount of residual red blood cells does not affect subsequent testing.

3. Cell Washing: Add over 5 volumes of PBS, HBSS, physiological saline, or serum-free medium to resuspend the cell precipitate, centrifuge at 4°C and 500 × g for 2-3 min, and discard the supernatant. Repeat this step once.

4. Resuspend and Count Cells: Resuspend the cell precipitate in an appropriate solution according to experimental requirements and count for subsequent experiments.

For Tissue Cell Samples (No Washing Required)

1. Cell Collection: Fresh tissues are digested with collagenase, trypsin, or similar enzymes and dispersed into a cell suspension, and centrifuged to discard the supernatant.

2. Cell Lysis: Dilute Red Blood Cell Lysis Buffer (10×) with deionized water to 1× concentration, add 5 volumes of 1× Lysis Buffer to resuspend the cells, and lyse on ice or at room temperature for 1-2 min. Add 15-20 mL PBS, HBSS, physiological saline, or serum-free medium and mix well. Centrifuge at 4°C and 500 × g for 5 min, and discard the red supernatant.

Note: If incomplete lysis of red blood cells is observed, repeat the lysis step once.

3. Resuspend and Count Cells: Resuspend the cell precipitate in an appropriate solution according to experimental requirements and count for subsequent experiments.

For Blood Samples (Requiring Washing)

1. Cell Collection: Collect fresh anticoagulated blood, centrifuge at 500 × g for 5 min, and discard the supernatant.

2. Cell Lysis: Dilute Red Blood Cell Lysis Buffer (10×) with deionized water to 1× concentration, add 6-10 volumes of 1× Lysis Buffer to resuspend the cells, and lyse on ice or at room temperature for 1-2 min. Centrifuge at 4°C and 500 × g for 5 min, and discard the red supernatant.

3. Cell Washing: Add over 5 volumes of PBS, HBSS, physiological saline, or serum-free medium to resuspend the cell precipitate, centrifuge at 4°C and 500 × g for 2-3 min, and discard the supernatant. Repeat this step once.

4. Resuspend and Count Cells: Resuspend the cell precipitate in an appropriate solution and count for subsequent experiments.

Note: Lysis time varies among blood sources. Mouse blood may require only 1-2 min, whereas human peripheral blood may require 4-5 min. Manually mix during lysis. For small blood samples, the first collection step may be omitted and 10 volumes of 1× Lysis Buffer can be added directly; mouse blood may be lysed for 4-5 min and human peripheral blood for up to 10 min, but not longer than 15 min.

Blood Samples (No Washing Required)

1. Cell Lysis: Dilute Red Blood Cell Lysis Buffer (10×) with deionized water to 1× concentration, add 10 volumes of 1× Lysis Buffer to fresh anticoagulated blood, and lyse on ice or at room temperature for 1-2 min. Centrifuge at 4°C and 500 × g for 5 min, and discard the red supernatant.

Note: For mouse blood, 4-5 min of lysis is sufficient, whereas human peripheral blood may require 10 min, but not more than 15 min. Manually mix during lysis.

2. Wash Cells: Add 20-30 mL PBS, HBSS, physiological saline, or serum-free culture medium and mix well. Centrifuge at 4°C and 500 × g for 5 min and discard the red supernatant. Repeat lysis if residual red blood cells remain.

3. Resuspend and Count Cells: Resuspend the cell precipitate in an appropriate solution according to experimental requirements and count for subsequent experiments.