Product Introduction
In the field of biological research, it is often necessary to remove red blood cells. There are various methods for red blood cell removal, such as ACK Lysis Buffer, Tris-ammonium chloride red blood cell lysis buffer, and Gey's Lysis Buffer. ACK Red Blood Cell Lysis Buffer, also known as ACK Lysis Buffer, is a solution used to lyse and remove enucleated red blood cells from tissue samples or blood of humans, mice, or other mammals. Its main active component is NH₄Cl (ammonium chloride).
The formula of ACK Lysis Buffer has been optimized. It can lyse enucleated red blood cells while causing almost no damage to lymphocytes or other nucleated cells. However, it has poor efficacy in lysing and removing nucleated red blood cells, such as those from birds or poultry, and is not recommended for use with such cells. This lysis buffer is sterilized by filtration. Blood or tissue cell samples treated with ACK Lysis Buffer can be used for subsequent experiments including cell culture, cell fusion, nucleic acid or protein extraction, as well as various routine analyses and detections. This reagent is for research use only and not intended for clinical diagnosis or any other purposes.
Materials to Be Prepared by Users
1. Trypsin, PBS (Phosphate Buffered Saline), HBSS (Hank's Balanced Salt Solution), normal saline or serum-free culture medium
2. Centrifuge
Operating Procedures (For Reference Only)
(1) Routine Operation for Tissue Cell Samples
1. Prepare cell suspension: Digest fresh tissues with trypsin, collagenase, or other suitable enzymes, prepare a cell suspension using appropriate methods, and centrifuge to discard the supernatant.
2. Lysis: Add 3-5 times the volume of the cell pellet of ACK Lysis Buffer, gently pipette to mix well, and lyse for 1-2 minutes. This step works better when performed at 4℃, but can also be carried out at room temperature.
3. Centrifugation: Centrifuge at 400-500×g for 5 minutes at 4℃, and discard the red supernatant. If a low-temperature centrifuge is not available, this step can also be performed at room temperature.
4. If red blood cell lysis is incomplete, repeat steps 2 and 3 once each.
5. Washing: Add an appropriate amount of PBS, HBSS, normal saline, or serum-free culture medium according to experimental requirements, gently mix to resuspend the pellet, and centrifuge at 400-500×g for 2-3 minutes at 4℃. Discard the supernatant. This centrifugation step can also be performed at room temperature. The volume of added PBS, HBSS, normal saline, or serum-free culture medium should generally be more than 5 times the volume of the cell pellet.
6. If necessary, repeat step 5 above for 1-2 washes in total.
7. Resuspend the cell pellet with an appropriate solution according to experimental needs, and proceed with subsequent experiments such as cell counting and culture.
(2) Rapid Operation for Tissue Cell Samples (No Washing Required)
1. Prepare cell suspension: Digest fresh tissues with trypsin, collagenase, or other suitable enzymes, prepare a cell suspension using appropriate methods, and centrifuge to discard the supernatant.
2. Lysis: Add 5 times the volume of the cell pellet of ACK Lysis Buffer, gently pipette to mix well, and lyse for 1-2 minutes. This step works better when performed at 4℃, but can also be carried out at room temperature.
3. Add 15-20 mL of PBS, HBSS, normal saline, or serum-free culture medium, and gently mix well.
4. Centrifugation: Centrifuge at 400-500×g for 5 minutes at 4℃, and discard the red supernatant. This centrifugation step can also be performed at room temperature.
5. If red blood cell lysis is incomplete, repeat steps 2-4 once each.
6. Resuspend the cell pellet with an appropriate solution according to experimental needs, and proceed with subsequent experiments such as cell counting and culture.
(3) Routine Operation for Blood Samples
1. Take fresh anticoagulated blood, centrifuge at 400-500×g for 5 minutes, and discard the supernatant.
2. Lysis: Add 6-10 times the volume of the cell pellet of ACK Lysis Buffer, gently pipette to mix well, and lyse for 1-5 minutes. This step works better when performed at 4℃, but can also be carried out at room temperature.
Important Note: For mouse blood, 1-2 minutes of lysis is sufficient. For human peripheral blood, it is advisable to extend the lysis time to 4-5 minutes and gently shake during lysis to facilitate red blood cell lysis.
3. Centrifugation: Centrifuge at 400-500×g for 5 minutes at 4℃, and discard the red supernatant. If a low-temperature centrifuge is not available, this step can also be performed at room temperature.
4. If red blood cell lysis is incomplete, repeat steps 2 and 3 once each.
5. Washing: Add an appropriate amount of PBS, HBSS, normal saline, or serum-free culture medium according to experimental requirements, gently mix to resuspend the pellet. Centrifuge at 400-500×g for 2-3 minutes at 4℃, and discard the supernatant. This centrifugation step can also be performed at room temperature. The volume of added PBS, HBSS, normal saline, or serum-free culture medium should generally be more than 5 times the volume of the cell pellet.
6. Resuspend the cell pellet with an appropriate solution according to experimental needs, and proceed with subsequent experiments such as cell counting and culture.
Note: For trace or small-volume blood samples, step 1 can be omitted. Directly add 10 times the blood volume of ACK Lysis Buffer and proceed to step 2, lysing at 4℃ or room temperature for 4-15 minutes. For mouse blood, 4-5 minutes of lysis is sufficient. For human peripheral blood, it is advisable to extend the lysis time to 10 minutes, but generally not more than 15 minutes, and the sample should be shaken appropriately during lysis to facilitate red blood cell lysis.
(4) Rapid Operation for Blood Samples (No Washing Required)
1. Add 10 times the volume of ACK Lysis Buffer to fresh anticoagulated blood, gently pipette to mix well, and lyse for 4-15 minutes. This step works better when performed at 4℃, but can also be carried out at room temperature.
Important Note: For mouse blood, 4-5 minutes of lysis is sufficient. For human peripheral blood, it is advisable to extend the lysis time to 10 minutes, but generally not more than 15 minutes, and the sample should be shaken appropriately during lysis to facilitate red blood cell lysis.
2. Add 20-30 mL of PBS, HBSS, normal saline, or serum-free culture medium, and gently mix well.
3. Centrifuge at 400-500×g for 5 minutes, and discard the red supernatant. Centrifugation at 4℃ yields better results.
4. If red blood cell lysis is incomplete, repeat steps 1-3 once.
5. Resuspend the cell pellet with an appropriate solution according to experimental needs, and proceed with subsequent experiments such as cell counting and culture.
Precautions
1. When preparing the cell suspension, it is not necessary to prepare a single-cell suspension unless required by the experiment.
2. If the subsequent experiment involves cell culture, sterile operation should be strictly followed, and the process should be conducted in a clean bench as much as possible.
3. Centrifugation steps should be performed in a 4℃ centrifuge whenever possible.
4. The difference between the routine procedure and the rapid procedure is as follows: the routine procedure includes an additional centrifugation step during washing, which can save the amount of washing solution, achieve better washing effect, and does not require large-volume centrifuge tubes; the rapid procedure omits one centrifugation step, resulting in a slightly poorer washing effect and requiring large-volume centrifuge tubes.
5. After centrifugation and washing, trace amounts of residual red blood cells usually do not affect subsequent detection.
6. If the samples treated with ACK Lysis Buffer are to be used for total RNA extraction, it is not necessary to use DEPC-treated solutions during the operation, i.e., there is no need to specifically remove RNase in this process.
7. Please use the reagent as soon as possible after opening to avoid affecting the results of subsequent experiments.
8. For your safety and health, please wear a lab coat and disposable gloves during operation.
Appendix (Calculation Formula for Converting Rotation Speed (rpm) to Centrifugal Force (×g))
g = r × 11.18 × 10⁻⁶ × rpm²Where:r = effective centrifugal radius, i.e., the distance from the center of the centrifuge rotor to the bottom of the centrifuge tube, in centimeters (cm).
Example: When the rotation speed is 3000 rpm and the effective centrifugal radius is 10 cm, the centrifugal force is calculated as follows:10 × 11.18 × 10⁻⁶ × 3000² = 1006.2 (×g)
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