Product Introduction
In the field of biological research, red blood cell removal is frequently required. 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. Gey's Lysis Buffer is a solution used to lyse and remove enucleated red blood cells from tissue samples or blood of humans, mice, other mammals, etc. It is mainly composed of phosphate, glucose, NH4Cl, and other ingredients.
The formula of Gey's Lysis Buffer has been optimized, containing carbonate, Ca²⁺, and Mg²⁺. It can lyse enucleated red blood cells while causing almost no damage to lymphocytes or other nucleated cells. This lysis buffer is sterilized by autoclaving and filtration. Blood or tissue cell samples treated with Gey's 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. It is especially suitable for removing red blood cells from skin cells and splenocytes. This reagent is for research use only, not for clinical diagnosis or any other purposes.
Materials to Be Prepared by Users
1. Trypsin, fetal calf serum (FCS), Hanks' balanced salt solution (HBSS)
2. Low-temperature centrifuge
Operating Procedures (for reference only)
(1) For Tissue Cell Samples
1. Prepare cell suspension: Digest fresh tissues with trypsin or collagenase, prepare a cell suspension using an appropriate method, and centrifuge to discard the supernatant.
2. Lysis: Add Gey's Lysis Buffer at a volume 5 times that of the cell pellet, gently pipette to mix well, and immediately place on ice for lysis for 5 minutes.
3. Add 100% FCS, centrifuge at 300g for 10 minutes at 4℃, and discard the red supernatant. This step can also be performed at room temperature if a low-temperature centrifuge is not available.
4. If red blood cell lysis is incomplete, repeat steps 2 and 3 once each.
5. Washing: Add an appropriate amount of HBSS according to experimental requirements, gently mix to resuspend the pellet, centrifuge at 400-500g for 2-3 minutes at 4℃, and discard the supernatant. This centrifugation step can also be performed at room temperature. The volume of HBSS added should generally be more than 5 times that of the cell pellet.
6. If necessary, repeat step 5 once, with a total of 1-2 washing cycles.
7. Resuspend the cell pellet with an appropriate solution according to experimental needs, and proceed to subsequent experiments such as cell counting and culture.
(2) For Blood Samples
1. Take fresh anticoagulated blood, centrifuge at 400-500g for 5 minutes, and discard the supernatant.
2. Lysis: Add Gey's Lysis Buffer at a volume 6-10 times that of the cell pellet, gently pipette to mix well, and immediately place on ice for lysis for 5 minutes.
Important Note: For mouse blood, 5 minutes of lysis is sufficient. For human peripheral blood, the lysis time should be extended to 5-10 minutes, and the mixture should be gently shaken during lysis to facilitate red blood cell lysis.
3. Add 100% FCS, centrifuge at 300g for 10-15 minutes at 4℃, and discard the red supernatant. This step can also be performed at room temperature if a low-temperature centrifuge is not available.
4. If red blood cell lysis is incomplete, repeat steps 2 and 3 once.
5. Washing: Add an appropriate amount of HBSS according to experimental requirements, gently mix to resuspend the pellet, centrifuge at 400-500g for 2-3 minutes at 4℃, and discard the supernatant. This centrifugation step can also be performed at room temperature. The volume of HBSS added should generally be more than 5 times that of the cell pellet.
6. Resuspend the cell pellet with an appropriate solution according to experimental needs, and proceed to subsequent experiments such as cell counting and culture.
Note: For trace or small-volume blood samples, step 1 can be omitted. Directly add Gey's Lysis Buffer at a volume 10 times that of the blood and proceed to step 2, with lysis performed at 4℃ for 10-15 minutes. For mouse blood, 15 minutes of lysis is sufficient; for human peripheral blood, the lysis time should be extended to 20 minutes, but generally should not exceed 20 minutes. The mixture should be shaken appropriately during lysis to promote red blood cell lysis.
Precautions
1. When preparing the cell suspension, it is not necessary to prepare a single-cell suspension unless required by the experiment.
2. If subsequent experiments involve cell culture, sterile techniques should be strictly followed, and operations should be performed in a clean bench as much as possible.
3. Centrifugation steps should be preferably carried out using a 4℃ centrifuge.
4. After centrifugation and washing, trace amounts of residual red blood cells usually do not affect subsequent detection.
5. If samples treated with Gey's Lysis Buffer are used for total RNA extraction, there is no need to use DEPC-treated solutions during cell processing, i.e., RNase removal is not required in this operation.
6. For your safety and health, wear a lab coat and disposable gloves during operation.
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