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（1） The principle is that when a mixed mononuclear cell suspension passes through nylon hair columns, B cells, plasma cells, monocytes, and some helper cells are selectively adhered to the nylon hair, while most T cells are obtained through nylon hair columns, which is an effective method for obtaining a rich T cell population.
（2） Materials and reagents
1. Femwall Laboratories, LP-1 Leukoqak Leukocell Filters.
2. Beaker, aluminum foil, funnel, disposable gloves, etc.
3. Fill the nylon bristle column with a disposable syringe.
4. Take the upper layer of naturally settling plasma and the layer of mononuclear cells between the obtained plasma and the layer after passing through the sucrose meglumine diatrizoate layer solution.
（3） Operation method
1. Cleaning and drying of nylon wool
(1) Wear disposable gloves that have washed away talcum powder, place nylon wool (1 or 2 bags, 35g each) into a beaker, add distilled or deionized water, cover the beaker with aluminum foil, and boil for about 10 minutes.
(2) Cool to room temperature and pour into a funnel to let the water drip dry.
(3) Repeat steps (1) and (2) 6 times.
(4) Spread the nylon wool on a square plate covered with gauze, dry it in a 37 ℃ oven for 2-3 days, and store it in a covered square plate.
2. Install nylon hair column
(1) Take a 50ml glass syringe, remove the injection core, and place a section of rubber tube with a clip on the syringe head.
(2) Sort out the nylon wool and fold it appropriately to fit the diameter of the syringe. Fill the syringe with approximately 20ml of volume.
(3) Wrap the syringe filled with nylon wool together with the syringe core and sterilize it under high pressure.
3. Cell separation
(1) Fix the syringe on the bracket, pour in 37 ℃ cell culture medium, close the valve for a certain period of time, then open the valve, drain the cell culture medium, clean the nylon wool several times, and close the valve.
(2) Dilute the cell fluid to be separated with pre heated culture medium to an appropriate concentration of approximately 5.00 × 107 cells/ml.
(3) Pour the cell fluid into the syringe so that it does not pass through the nylon hair column. Cover the syringe and incubate at 37 ℃ for 45 minutes to 1 hour.
(4) Open the lower opening, slowly release (1 drop/min), and collect in a centrifuge tube.
(5) Centrifuge to obtain the required T lymphocytes.
(6) Close the lower opening of the syringe, add 0.85% cold physiological saline to the syringe, shake, and cover the syringe core. Open the lower opening and vigorously push out the liquid inside the syringe to obtain B lymphocytes, plasma cells, macrophages, etc. that adhere to the nylon hair.
（4） Precautions
1. In this separation method, a portion of T lymphocytes is often adsorbed, and the amount of adsorption is related to the quality of nylon wool and the tightness of the column.
2.The recovery rate of T lymphocytes in this method is about 20% to 30%.
3. The used nylon wool can be recycled, washed with salt water, then immersed in 0.1Mol/L HCl overnight, and then cleaned using the same method as before. Monocyte isolation technology.
（1） Iron powder adsorption method
(1) Iron powder or carbonyl iron powder (Atomergic metals) has a purity of 99% and particles smaller than 60 µ m (Goodfellow Metals).
(2) Strong magnet (horseshoe shaped).
(3) A small rod-shaped magnet (approximately 1cm long).
(4) Capillary straws, etc.
2. Operation method
(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 out salt water, use a strong magnet to absorb the iron powder.
(2) Suspend iron powder with 50ml of physiological saline. After shaking well, divide into 10 bottles, wrap the bottle mouth, sterilize at 121 ℃ for 20 minutes, and immediately shake gently to prevent iron powder from clumping. Store for future use.
(3) Before use, remove the physiological saline from the bottle and add an appropriate amount of mononuclear cell suspension (3ml~5ml, with a total cell count of 8) × 107 pieces/bottle). Incubate at 37 ℃ for 45 minutes, occasionally shaking in the middle to suspend the iron powder.
(4) Add a small magnet rod to the bottle and let it absorb the iron powder and the cells attached to it.
(5) Pour out the suspension, which is mainly composed of lymphocytes, centrifuge, and collect.
(6) Pour a certain amount of Hank's liquid into the iron powder bottle, shake it vigorously, and adsorb it with a strong magnet. After a few minutes, pour out the liquid.
(7) Centrifuge at 2 000r/min with monocytes at the bottom of the tube.
（2） The glass plate adsorption method tilts the separated mononuclear cells into a sterile and clean glass plate, and places them at 37 ℃ for 30-40 minutes. The suspension is gently aspirated using a capillary pipette to form lymphocytes. Rinse the plate with an appropriate amount of Hank's solution, and the harvested monocyte suspension is obtained.