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Grouping	Other reagents
Keywords	Red Blood Cell Lysis Buffer
Brand	NJDULY
Place of Origin	Nanjing, China (Mainland)
Payment	T/T
Port	Shanghai
Supply Capacity	100  Piece/per Month
Packing	25g/piece
Other packaging	please consult customer service
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Product introduction
Red blood cell lysis buffer, also known as ack lysis buffer, is a solution used to lyse and remove non nuclear red blood cells from human or rat blood or tissue samples.
The lysate was optimized to lyse red blood cells without damaging lymphocytes or other cells with nuclei.
The main effective component of the pyrolysis liquid is ammonium chloride.
This lysate is not suitable for the lysis of erythrocytes with nuclei, such as erythrocytes of birds or birds.
After aseptic treatment, the treated blood or tissue cell samples can be used for subsequent primary culture, cell fusion, nucleic acid or protein extraction and various conventional analysis and detection.
instructions
For tissue and cell samples:
1. The fresh tissue was digested by collagenase or trypsin, and then dispersed into cell suspension by appropriate methods, and the supernatant was discarded by centrifugation.
2. Add 3-5 times of cell volume of red blood cell lysate, gently blow and mix, and lyse for 1-2 minutes. For example, if the volume of cell precipitation is 1 ml, 3-5 ml of erythrocyte lysate is added. This step can be operated at room temperature or 4 ℃.
3. Centrifugation at 400-500g for 5 minutes, discard the red supernatant. 4 ℃ centrifugation is better.
4. If it is found that the red blood cell lysis is incomplete, the above steps 2 and 3 can be repeated once. Usually, a very small amount of red blood cells will not affect some subsequent tests.
5. Wash 1-2 times: add appropriate amount of PBS, HbSS, normal saline or serum-free culture medium, suspend and precipitate again, centrifuge 400-500g for 2-3min, and discard the supernatant. It can be repeated for 1-2 times. The amount of detergent should be at least 5 times the volume of cell precipitation. 4 ℃ centrifugation is better.
6. According to the needs of the experiment, the cells can be suspended with appropriate solution and then counted.
For tissue and cell samples, the rapid operation steps without washing are as follows:
1. The fresh tissue was digested by collagenase or trypsin, and then dispersed into cell suspension by appropriate methods, and the supernatant was discarded by centrifugation.
2. For 0.2ml of cell precipitation, add 1ml of erythrocyte lysate, gently blow and mix, and lyse for 1-2 minutes. This step can be operated at room temperature or 4 ℃.
3. Add 15-20ml PBS, HbSS, normal saline or serum-free medium and mix well.
4. Centrifugation at 400-500g for 5 minutes, discard the red supernatant. 4 ℃ centrifugation is better.
5. If it is found that the red blood cell lysis is incomplete, the above steps 2 and 3 can be repeated once. Usually, a very small amount of red blood cells will not affect some subsequent tests.
6. According to the needs of the experiment, the cells can be suspended with appropriate solution and then counted.
Note: for the conventional step, one more step of centrifugation in the washing process can save the amount of washing liquid, and the washing effect is better, at the same time, it does not need a large centrifugal tube. The rapid step is less than one centrifugation, but the washing effect is slightly worse, and a large centrifugal tube is needed.
For blood samples:
1. Take fresh anticoagulant, centrifuge 400-500g for 5min, and discard the supernatant.
2. Add 6-10 times of cell volume of red blood cell lysate, gently blow and mix, and lyse for 1-2 minutes. For example, if the volume of cell precipitation is 1 ml, 6-10 ml of erythrocyte lysate is added. This step can be operated at room temperature or 4 ℃. Note: for rat blood, 1-2 minutes is enough, for human peripheral blood, it is better to extend the lysis time to 4-5 minutes, and shake occasionally to promote the lysis of red blood cells.
3. Centrifugation at 400-500g for 5 minutes, discard the red supernatant. 4 ℃ centrifugation is better.
4. If it is found that the red blood cell lysis is incomplete, the above steps 2 and 3 can be repeated once. Usually, a very small amount of red blood cells will not affect some subsequent tests.
5. Wash 1-2 times: add appropriate amount of PBS, HbSS, normal saline or serum-free culture medium, suspend and precipitate again, centrifuge 400-500g for 2-3min, and discard the supernatant. It can be repeated for 1-2 times. The amount of detergent should be at least 5 times the volume of cell precipitation. 4 ℃ centrifugation is better.
6. According to the needs of the experiment, the cells can be suspended with appropriate solution and then counted.
Note: for micro or small amount of blood samples, it is not necessary to centrifuge and discard the supernatant in the first step, but directly add 10 times of the volume of blood in the second step, and lyse at room temperature or 4 ℃ for 4-5 minutes. For mouse blood, 4-5 minutes is enough. For human peripheral blood, the lysis time should be extended to 10 minutes, but usually not more than 15 minutes. In addition, it should be shaken occasionally to promote the lysis of red blood cells. The following steps are the same.
For the rapid operation steps of blood sample without washing:
1. Add 10ml of red blood cell lysate into every 1ml of fresh anticoagulant, gently blow and mix, and lyse for 4-5 minutes. This step can be operated at room temperature or 4 ℃. Note: for rat blood, 4-5 minutes is enough. For human peripheral blood, the lysis time should be extended to 10 minutes, but usually not more than 15 minutes. In addition, it should be shaken occasionally to promote the lysis of red blood cells.
2. Add 20-30ml PBS, HbSS, normal saline or serum-free medium and mix well.
3. Centrifugation at 400-500g for 5 minutes, discard the red supernatant. 4 ℃ centrifugation is better.
4. If it is found that the red blood cell lysis is incomplete, the above steps 2 and 3 can be repeated once. Usually, a very small amount of red blood cells will not affect some subsequent tests.
5. According to the needs of the experiment, the cells can be resuspended with appropriate solution and then counted.
Note: for the conventional step, one more step washing process of centrifugation, but can save the amount of detergent, and the washing effect is also better, at the same time does not need a large volume of centrifugal tube. The rapid step is less than one centrifugation, but the washing effect is slightly worse, and a large centrifugal tube is needed.
matters needing attention
1. The preparation of cell suspension should be based on the needs of specific experiments, and it is not necessary to prepare single cell suspension.
2. If the follow-up test is used for cell culture, attention should be paid to aseptic operation in the process of operation, and the operation should be carried out in the ultra clean workbench as far as possible.
3. The centrifugation step should be operated on a 4 ℃ centrifuge as far as possible.
4. The difference between the conventional step and the fast step is: the conventional step has one more step of centrifugation in the washing process, which can save the amount of washing liquid, and the washing effect is also better, and it does not need a large centrifugal tube; the fast step has one less centrifugal process, and the washing effect is slightly worse, and it also needs a large centrifugal tube.
5. After centrifugation and washing, usually a very small amount of red blood cells will not affect the subsequent detection.
6. If the sample treated by ack lysis buffer is used for the extraction of total RNA, there is no need to use DEPC solution, that is, there is no need to remove RNase in this operation.
7. For your safety and health, please wear lab clothes and disposable gloves.