(1) Principle When mixing a mononuclear cell suspension through a nylon hair column, B cells, plasma cells, monocytes and some auxiliary cells are selectively adhered to the nylon hair, while most T cells pass through the nylon hair column This is an effective method to obtain T-cell-rich populations.
(2) Materials and reagents
1. Nylon Wool Fiber.
2. Beaker, aluminum foil, funnel, disposable gloves, etc.
3. Filled with nylon wool columns, disposable syringes.
4. After taking the naturally sedimented upper plasma and passing through the sucrose-Pantrime meglumine stratified liquid, the mononuclear cell layer between the obtained plasma and the stratified liquid is obtained.
(3) Operation method
1. Washing and drying of nylon wool (1) Put on disposable gloves that have been washed with talc powder, put nylon wool (1 pack or 2 packs, 35g each pack) into a beaker, add distilled or deionized water, and cover with aluminum foil Put on the beaker and boil for about 10min.
(2) Cool to normal temperature, pour into the funnel, and let the water drop dry.
(3) Repeat steps (1) and (2) 6 times.
(4) Spread the nylon wool in a square dish covered with gauze, dry it for 2 to 3 days in a 37 Â° C incubator, and store it in a square dish with a lid.
2. Install a nylon wool column (1) Take a 50ml glass syringe, pull out the injection core, and put a piece of rubber tube with a clip on the syringe head.
(2) Comb the nylon wool and fold it properly to fit the diameter of the syringe and fill the syringe with a volume of about 20ml.
(3) Wrap the syringe filled with nylon wool together with the syringe core and autoclave.
3. Cell separation (1) Fix the syringe on the holder, pour the cell culture solution at 37 â„ƒ, close the valve for a certain period of time, then open the valve, let go of the cell culture solution, wash the nylon hair several times, and close the valve.
(2) Dilute the cell liquid to be separated into an appropriate concentration with pre-warmed culture liquid, about 5.00 Ã— 107 cells / ml.
(3) Pour the cell fluid into the syringe so that it does not pass the nylon wool column. Cover the syringe and incubate at 37 Â° C for 45min
(4) Open the lower mouth, slowly drain (1 drop / min), and collect in a centrifuge tube.
(5) Centrifuge to obtain the required T lymphocytes.
(6) Close the lower mouth of the syringe, add 0.85% ice-cold physiological saline into the syringe, oscillate, and put on the syringe core, open the lower mouth, and push out the liquid in the syringe, to obtain B lymphocytes and pulp adhered to the nylon wool Cells, macrophages, etc.
(Four) matters needing attention
1. In this separation method, a part of T lymphocytes are often adsorbed. The amount of adsorption is related to the quality of nylon wool and the tightness of the packed column.
2. The recovery rate of T lymphocytes in this method is about 20% to 30%.
3. Used nylon wool can be recovered, washed with brine, and then immersed in 0.1 Mol / L HCl overnight, and then washed in the same way as before.
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