Red blood cell lysate (RBC Lysis Buffer, 10×) instruction manual

Red blood cell lysate (RBC Lysis Buffer, 10×)

Introduction

In the field of biological research, it is often necessary to remove red blood cells. There are many ways to remove red blood cells, such as ACK Lysis Buffer, Tris-ammonium chloride red blood cell lysate, and Gey's Lysis Buffer. Red Blood Cell Lysis Buffer is a solution for lysing and removing non-nuclear red blood cells from tissue samples or blood in human, mouse or other mammals, and its main active ingredient is NH4CI.

The RBC Lysis Buffer (10×) formulation is optimized to differ from ACK Lysis Buffer in that it cleaves seedless red blood cells with little damage to Lymphocytes or other nucleated cells. It is not recommended for lysis and removal of red blood cells with nucleated red blood cells, such as birds or birds. It is not recommended when lysing similar cells. The lysis is filtered and sterilized as a 10× concentrate. Blood or tissue cell samples treated with 1×RBC Lysis Buffer can be used for subsequent cell culture, cell fusion, nucleic acid or protein extraction and various routine analyses. And detection, especially for flow cytometry or where high concentrations of lysate are required.

composition:

Bring your own materials:

1. Trypsin
2. Centrifuge
3. PBS, HBSS, saline or serum-free medium

Operation steps (for reference only):

Note: In most cases, RBC Lysis Buffer (10×) should be diluted to 1× using sterile deionized water, except for flow cytometry.

• Conventional operation of tissue cell samples

1. Preparation of cell suspension: Fresh tissue is digested with trypsin or collagenase, prepared into a cell suspension by an appropriate method, and the supernatant is discarded by centrifugation.
2. Lysis: Add 3×5 times the cell pellet volume of 1×RBC Lysis Buffer, mix gently and lyse for 1~2 min.

This operation is at 4. It is better to operate under C conditions and can also be operated at room temperature.

1. Centrifugation: 4. C, 400~500g was centrifuged for 5 min, and the red supernatant was discarded. This step can also be operated at room temperature.
2. If red blood cell lysis is found to be incomplete, repeat steps 2 and 3 above.
3. Washing: Add appropriate amount of PBS, HBSS, normal saline or serum-free medium according to the experimental requirements, and gently resuspend the pellet. 4. C, 400~500g is centrifuged for 2~3 minutes, the supernatant is discarded, and the centrifugation step can also be operated at room temperature. The amount of PBS, HBSS, physiological saline or serum-free medium to be added should generally be more than 5 times the volume of the cell pellet.
4. If necessary, repeat step 5 above for a total of 1 or 2 washes.
5. The cell pellet was resuspended in an appropriate solution according to the experiment, and subsequent experiments such as counting and culturing were performed.

• Rapid manipulation of tissue cell samples (no washing required)

1. Preparation of cell suspension: Fresh tissue is digested with trypsin or collagenase to prepare a cell suspension, and the supernatant is discarded by centrifugation.
2. Lysis: Add 1×RBC Lysis Buffer with 5 times cell pellet volume of the cells, mix gently and lyse for 1~2 min. This operation is at 4. It is better to operate under C conditions and can also be operated at room temperature.
3. Add 15~20 ml PBS, HBSS, normal saline or serum-free medium and mix gently.
4. Centrifugation: 4. C, 400~500g is centrifuged for 5 min, the red supernatant is discarded, and the centrifugation step can also be operated at room temperature.
5. If red blood cell lysis is found to be incomplete, repeat steps 2 through 3 above.
6. The cell pellet was resuspended in an appropriate solution according to the experiment, and subsequent experiments such as counting and culturing were performed.

• Routine operation of blood samples

1. Take fresh anticoagulation, centrifuge at 400~500 g for 5 min, discard the supernatant.
2. Lysis: Add 6×10 times the cell pellet volume of 1×RBC Lysis Buffer, gently mix and mix, and lyse for 1~5min.

This operation is at 4. It is better to operate under C conditions and can also be operated at room temperature. (Special reminder: For the blood of rats, lysis for 1~2 min is enough. For human peripheral blood, it is advisable to prolong the lysis time to 4~5 min, and gently shake during the lysis process to promote erythrocyte lysis.)

1. Centrifugation: 4. C, 400~500g is centrifuged for 5 min, the red supernatant is discarded, and the centrifugation step can also be operated at room temperature.
2. If red blood cell lysis is found to be incomplete, repeat steps 2 and 3 above.
3. Washing: Add appropriate amount of PBS, HBSS, normal saline or serum-free medium according to the experimental requirements, and gently resuspend the pellet. 4. C, 400~500g is centrifuged for 2~3 minutes, the supernatant is discarded, and the centrifugation step can also be operated at room temperature. The amount of PBS, HBSS, physiological saline or serum-free medium to be added should generally be more than 5 times the volume of the cell pellet.
4. The cell pellet was resuspended in an appropriate solution according to the experiment, and subsequent experiments such as counting and culturing were performed.

Note: For a small or small amount of blood sample, you can use the ACK Lysis Buffer of 10 times the blood volume without the first step, and proceed to step 2, and at 4. C or room temperature cleavage for 4 to 15 min. For the blood of rats, lysis for 4 to 5 minutes is sufficient. For human peripheral blood, the lysis time should be extended to 10 min, but usually should not exceed 15 min, and the lysis process should be properly shaken to promote erythrocyte lysis.

• Quick operation of blood samples (no washing required)

1. Add 10 volumes of 1× RBC Lysis Buffer to fresh anticoagulant, mix gently by pipetting, or operate at room temperature. (Special reminder: For the blood of rats, lysis for 4~5 minutes is enough. For human peripheral blood, it is advisable to prolong the lysis time to 10 min, but usually should not exceed 15 min, and the lysis process should be properly shaken to promote erythrocyte lysis.)
2. Add 20~30 ml PBS, HBSS, normal saline or serum-free medium and mix gently.
3. If red blood cell lysis is found to be incomplete, repeat steps 2 and 3 above.
4. The cell pellet was resuspended in an appropriate solution according to the experiment, and subsequent experiments such as counting and culturing were performed.

Precautions:

1. The preparation of the cell suspension should be carried out according to the needs of the experiment, and it is not necessary to prepare a single cell suspension.
2. If the subsequent test is for cell culture, the aseptic operation should be taken during the operation and try to operate in the ultra-clean workbench.
3. Centrifuge steps as far as possible at 4. C operation on the centrifuge.
4. The difference between the conventional step and the quick step is that the conventional step has one more centrifugation of the washing process, the amount of the washing liquid can be saved, and the washing effect is also better, and a large volume of the centrifuge tube is not required; the rapid step has one less centrifugal process. The washing effect is also better, and a large volume of the centrifuge tube is not required; the rapid step has one less centrifugal process, the washing effect is slightly worse, and a large volume of the centrifuge tube is required.
5. After centrifugation, usually a very small amount of red blood cells does not affect subsequent detection.
6. If the sample treated with 1×RBC Lysis Buffer is subsequently used for the extraction of total RNA, it is not necessary to use the DEPC-treated solution when processing the cells, ie, it is not necessary to intentionally remove the RNase in this operation.
7. For your safety and health, wear a lab coat and disposable gloves.

Validity: Valid for 12 months.

If you encounter any technical problems in the experiment, please consult the company's technical staff in time.

Amino Acids

Amino acid additives are used in feed to balance or supplement a particular production purpose of the required nutrients. The main amino acids added in the feed are: lysine, tryptophan, methionine, threonine these limiting amino acids. Amino acids are the basic building blocks of proteins. The essence of protein nutrition is amino acid nutrition. The core of amino acid nutrition is the balance between amino acids.

The balance of amino acids in natural feed is very poor. Almost all of them are not balanced. The animal body in the limited feed can not synthesize themselves.

They can only rely on eating intake of amino acids. The amino acid content of natural feed is very different, each is not the same, because of different kinds, different ratio of natural feed composed of full price compound feed, although try to mix according to the principle of amino acid balance, but their various amino acid content and the proportion between amino acid is still variable, various. Therefore, amino acid additives are needed to balance or supplement the requirements for a particular production purpose.

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