Red Blood Cell Lysis Buffer

Instructions:

For tissue cell samples:
1. Digest fresh tissue with collagenase or trypsin, disperse into a cell suspension using an appropriate method, and centrifuge to discard the supernatant.
2. Add 3-5 times the volume of red blood cell lysis buffer to the cell pellet, gently pipette to mix, and lyse for 1-2 minutes. For example, if the cell pellet volume is 1 ml, add 3-5 ml of red blood cell lysis buffer. This step can be performed at room temperature or 4°C.
3. Centrifuge at 400-500g for 5 minutes and discard the red supernatant. Centrifugation at 4°C is most effective.
4. If red blood cell lysis is incomplete, repeat steps 2 and 3 above. Generally, very small amounts of red blood cells will not affect subsequent testing. 5. Wash 1-2 times: Add an appropriate amount of PBS, HBSS, saline, or serum-free culture medium, resuspend the pellet, centrifuge at 400-500g for 2-3 minutes, and discard the supernatant. Repeat once more for a total of 1-2 washes. The amount of wash buffer used should generally be at least 5 times the volume of the cell pellet. Centrifugation at 4°C is optimal. 6. Resuspend the cell pellet in an appropriate solution based on experimental needs and proceed with counting and other subsequent experiments. Quick steps for tissue cell samples without washing: 1. Digest fresh tissue with collagenase or trypsin, disperse into a cell suspension using an appropriate method, and centrifuge to discard the supernatant. 2. Add 1 ml of red blood cell lysis buffer to 0.2 ml of cell pellet, gently pipette to mix, and lyse for 1-2 minutes. This step can be performed at room temperature or 4°C.
3. Add 15-20ml of PBS, HBSS, saline, or serum-free culture medium and mix well.
4. Centrifuge at 400-500g for 5 minutes and discard the red supernatant. Centrifugation at 4°C is optimal.
5. If incomplete lysis of red blood cells is detected, repeat steps 2 and 3. Typically, very small amounts of red blood cells will not affect subsequent assays.
6. Resuspend the cell pellet in an appropriate solution as needed for the experiment and proceed with counting and other subsequent assays.
Note: The conventional procedure includes an additional centrifugation step during the wash process, but this saves on wash buffer, improves wash efficiency, and eliminates the need for large centrifuge tubes. The rapid procedure eliminates the need for centrifugation, but the wash efficiency is slightly worse and requires larger centrifuge tubes.

For blood samples:
1. Centrifuge fresh anticoagulated blood at 400-500g for 5 minutes and discard the supernatant. 2. Add 6-10 times the volume of cell pellet to the lysing buffer, gently pipette to mix, and lyse for 1-2 minutes. For example, if the cell pellet volume is 1 ml, add 6-10 ml of lysing buffer. This step can be performed at room temperature or 4°C. Note: For mouse blood, lysis for 1-2 minutes is sufficient. For human peripheral blood, the lysis time should be extended to 4-5 minutes. Occasional shaking should be performed during lysis to promote lysis of the red blood cells. 3. Centrifuge at 400-500g for 5 minutes and discard the red supernatant. Centrifugation at 4°C is more effective. 4. If the red blood cells are not completely lysed, repeat steps 2 and 3 above. Usually, a very small amount of red blood cells will not affect subsequent assays. 5. Wash 1-2 times: Add an appropriate amount of PBS, HBSS, saline, or serum-free medium, resuspend the pellet, centrifuge at 400-500g for 2-3 minutes, and discard the supernatant. Repeat once more, for a total of 1-2 washes. The volume of wash buffer used should generally be at least 5 times the volume of the cell pellet. Centrifugation at 4°C is optimal.
6. Resuspend the cell pellet in an appropriate solution as needed for your experiment and proceed with counting and other subsequent experiments.
Note: For trace or small amounts of blood samples, skip centrifugation and discarding the supernatant in the first step. In the second step, add 10 times the volume of red blood cell lysis buffer and lyse the cells at room temperature or 4°C for 4-5 minutes. For mouse blood, 4-5 minutes is sufficient. For human peripheral blood, the lysis time should be extended to 10 minutes, but generally should not exceed 15 minutes. Occasional shaking should be performed during the lysis process to promote red blood cell lysis. Subsequent steps are the same.

Quick procedure for blood samples without washing:
1. Add 10 ml of red blood cell lysis buffer to every 1 ml of fresh anticoagulated blood, gently pipette to mix, and lyse for 4-5 minutes. This step can be performed at room temperature or 4°C. Note: For mouse blood, lysis for 4-5 minutes is sufficient. For human peripheral blood, the lysis time should be extended to 10 minutes, but generally should not exceed 15 minutes. Occasional shaking is recommended during the lysis process to promote red blood cell lysis. 2. Add 20-30ml of PBS, HBSS, saline, or serum-free culture medium and mix thoroughly. 3. Centrifuge at 400-500g for 5 minutes and discard the red supernatant. Centrifugation at 4°C is more effective. 4. If red blood cell lysis is incomplete, repeat steps 2 and 3 above. Usually, a very small amount of red blood cells will not affect subsequent assays. 5. Resuspend the cell pellet in an appropriate solution as needed for the experiment and proceed with counting and other subsequent experiments. Note: In the conventional procedure, the addition of a wash step (centrifugation) saves wash buffer, improves wash efficiency, and eliminates the need for large centrifuge tubes. The rapid step eliminates the need for centrifugation, but the washing effect is slightly worse and larger centrifuge tubes are required. Notes: 1. If samples treated with red blood cell lysis buffer are subsequently used for total RNA extraction, it is not necessary to use DEPC-treated solutions when handling the cells. 2. For your safety and well-being, please wear a lab coat and disposable gloves during operation. 3. This product is for professional research use only. It is not intended for clinical diagnosis or treatment, for use with food or medicine, or for storage in residential areas.