(I) Principles 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. It is an effective way to obtain a T-rich cell population. (2) Materials and reagents 1. Nylon hair (Femwall Laboratories, LP-1 Leukoqak leukocyte Filters). (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 or 2 packs, 35g per pack) into the beaker, add distilled or deionized water, cover the beaker with aluminum foil and boil for about 10min. . 2. 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. 3. Cell separation by centrifuge (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. (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 packed column. (1) Iron powder adsorption method 1. Materials and reagents (1) Iron powder or Atomergic chemetals is 99% pure and the particles are less than 60 μm (Goodfellow Metals). 2. Method of operation (1) Weigh a certain amount of iron powder, usually 10g, and wash it 4 times with 100ml of physiological saline to remove any soluble toxic substances. When pouring the brine, use a strong magnet to hold the iron powder. (2) Glass plate adsorption method The separated mononuclear cells are poured into a sterile clean glass plate, placed at 37 ° C for 30 min to 40 min, and the suspension is gently aspirated with a capillary pipette, which is a lymphocyte. Rinse the plate with an appropriate amount of Hank's solution and harvest as a mononuclear cell suspension. More mini centrifuge information login: Ningbo Siny Medical Technology Co., Ltd , https://www.sinymedical.com
2. Beakers, aluminum foil, funnels, disposable gloves, etc.
3. Load nylon wool column, disposable Syringe.
4. Take the naturally deposited upper layer of plasma, and after passing through the sucrose-diatrizoate stratified solution, obtain a mononuclear cell layer between the plasma and the stratified fluid.
(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) 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) Wrap the syringe filled with nylon wool together with the syringe core and autoclave.
(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. The syringe was capped and incubated at 37 ° C for 45 min to 1 h.
(4) Open the lower mouth and slowly discharge (1 drop/min) and collect in the 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.
2. The recovery rate of T lymphocytes in this method is about 20% to 30%.
3. The used nylon wool was recovered, washed with brine, then immersed in 0.1 Mol/L HCl overnight and then washed with the previous method. Monocyte separation technique
(2) Strong magnet (horsehoe shape).
(3) A small rod magnet (about 1 cm long).
(4) Capillary straws, etc.
(2) The iron powder was suspended in 50 ml of physiological saline. After shaking, dispense into 10 bottles, wrap the bottle mouth, sterilize at 121 °C for 20 min, shake it gently after taking out, prevent the iron powder from agglomerating and store for later use.
(3) Before use, pour the physiological saline in the bottle and add an appropriate amount of mononuclear cell suspension (3 ml to 5 ml, total number of cells is 8 × 107 cells/bottle). Incubate at 37 ° C for 45 min, shaking from time to time, so that the iron powder is suspended.
(4) Add a small magnet rod to the bottle to hold the iron powder and the cells attached to the iron powder.
(5) Pour the suspension, which is mainly lymphocytes, centrifuged, and collected.
(6) Pour a certain amount of Hank's solution into the iron powder bottle, shake it vigorously, then absorb it with a strong magnet. After a few minutes, pour out the liquid.
(7) Centrifugal liquid at 2 000 r/min, the bottom of which is a mononuclear cell.