Red blood cell lysis buffer is the simplest and most feasible method for removing red blood cells, which involves using lysis buffer to lyse red blood cells. It does not damage nucleated cells and can effectively remove red blood cells. Lysis is a relatively mild method for removing red blood cells, mainly used for the separation and purification of tissue cells dispersed by enzymatic digestion, the separation and purification of lymphocytes, and the removal of red blood cells in experiments such as tissue cell protein and nucleic acid extraction.
Principle explanation
By utilizing the difference in ion concentration between inside and outside the cell, an osmotic pressure difference is formed, and external water will diffuse into the cell, causing red blood cells to expand and achieve the effect of lysis.
usage method
1. Lysis of red blood cells
(1) Add 3 times the volume of red blood cell lysis buffer to 1 portion of fresh whole blood to be lysed (e.g. 1mL of fresh whole blood is added to 3mL of red blood cell lysis buffer)
Gently swirl or invert and mix well. [Note]: If fresh tissue cells need to be processed, they need to be digested with trypsin or other enzymes to form a single cell suspension and then centrifuged for collection
Collect cells before proceeding with subsequent operations.
(2) Incubate on ice for 15 minutes, gently vortex and mix twice during this time. [Note]: After red blood cell lysis, the solution should be clear and transparent.
2. Collect white blood cells
(1) Collect white blood cells by centrifugation at 450 × g for 10 minutes at 4 ℃, and carefully discard the supernatant.
(2) Add twice the volume of red blood cell lysis buffer to the above precipitate, gently vortex and resuspend white blood cells thoroughly (for example, if the initial blood volume is 1 mL, add
Add 2 mL of red blood cell lysate.
(3) Collect white blood cells by centrifugation at 450 × g for 10 minutes at 4 ℃, and carefully and thoroughly aspirate the supernatant.
(4) Resuspend cells for subsequent experiments. [Note]: If extracting RNA, it is best to start using a solution without RNase from this step.
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