(1) Principle
When a single nuclear cell suspension is passed through a nylon hair column, B cells, plasma cells, monocytes, and some helper cells are selectively attached to the nylon hair, while most T cells pass through the nylon hair column, which is rich. An effective method for containing T cell populations.
(2) Materials and reagents
1. Nylon hair (Femwall Laboratories, LP-1 Leukoqak leukocyte Filters).
2. Beakers, aluminum foil, funnels, disposable gloves, etc.
3. Fill the nylon hair column, disposable syringe.
4. Take the naturally deposited upper layer of plasma, after the sucrose-diazepam leaching solution, obtain a mononuclear cell layer between the plasma and the stratified fluid.
(3) Operation method
1. Cleaning and drying of nylon wool
(1) Put on disposable gloves that have been washed with talcum powder, put nylon wool (1 pack or 2 packs, 35g per pack) into the beaker, add distilled water or deionized water, cover the beaker with aluminum foil and boil for about 10min. .
(2) Cool to room temperature and pour into the funnel to allow the water to dry.
(3) Repeat steps (1) and (2) six times.
(4) Spread the nylon wool in a square plate covered with gauze, and dry it in a 37 ° C incubator for 2 to 3 days, then store it in a square plate with a lid.
2. Packed with nylon hair column
(1) Take a 50ml glass syringe, remove the injection core, and put a piece of rubber with a clip on the syringe head.
(2) The nylon hair is combed and appropriately folded to fit the diameter of the syringe, and filled into the syringe, about 20 ml in volume.
(3) The syringe filled with nylon hair is wrapped together with the syringe core and autoclaved.
3. Cell separation
(1) Fix the syringe on the holder, pour the cell culture solution at 37 ° C, close the valve for a certain time, then open the valve, let go of the cell culture solution, wash the nylon hair several times, and close the valve.
(2) The cell liquid to be separated is diluted with a pre-warmed culture solution to an appropriate concentration of about 5.00 x 107 cells/ml.
(3) Pour the cell liquid into the syringe so that it does not pass through the nylon hair column. Cover with syringe and incubate for 45 min at 37 °C
Until 1h.
(4) Open the lower mouth and slowly discharge (1 drop/min) and collect in a centrifuge tube.
(5) Centrifugation to obtain the desired T lymphocytes.
(6) Close the lower mouth of the syringe, add 0.85% ice-cold physiological saline to the syringe, shake it, put on the syringe core, open the lower mouth, and push out the liquid in the syringe to obtain the B lymphocytes and pulp adhering to the nylon hair. Cells, macrophages, etc.
(four) matters needing attention
1. In this separation method, T lymphocytes are often partially adsorbed, and the amount of adsorption is related to the quality of the nylon hair, and is also related to the tightness of the packing column.
2. The recovery rate of T lymphocytes in this method is about 20% to 30%.
3. The used nylon wool can be recovered, washed with salt water, then immersed in 0.1Mol/L HCl overnight, and then washed with the same method.
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