Panning separation method to separate T cell subsets

Panning separation method to separate T cell subsets

The polystyrene surface can be used as an insoluble matrix for affinity reagents to separate lymphocyte subpopulations. The polystyrene petri dish is first coated with purified goat anti-mouse IgG antibody, and then the cell suspension in which the mouse anti-human (such as anti-CD4 or anti-CD8 monoclonal antibody) reacts with T cells is added to the petri dish. After incubating for a period of time, T cells specific for CD4 or CD8 antigen bind to the surface of the plate, but CD8+ or CD4+ cells do not bind. Gently aspirate the non-adherent cell suspension and wash off the adherent cells.
[Reagents and preparation]
(1) Polysucrose-Vantoin meglumine layering solution (D=1.077±0.001)
(2) Anti-CD4 and anti-CD8 monoclonal antibodies
(3) Goat anti-mouse IgG antibody
(4) Hank’s solution (containing 5% calf serum, without Ca2+, Mg2+)
(5) pH 9.0, 0.05mol/L Tris-HCl buffer
(6) pH 7.5, 0.15mol/L PBS (containing 10% calf serum)
(7) pH 7.2, 0.15mol/L PBS
(8) Lidocaine hydrochloride (20mg/ml)

[Operation method]
(1) Goat anti-mouse IgG antibody coated polystyrene plate
① Prepare 10mg/ml goat anti-mouse IgG antibody solution with pH 9.0, 0.05mol/L Tris-HCl buffer;
②Pick an appropriate amount of antibody solution in a petri dish and incubate at room temperature for 1.5h or 4℃ for 18h;
③ Wash the petri dish 3 times with about 5ml of pH 7.5, 0.15mol/L PBS to remove unbound antibodies;
④Add 5ml of pH 7.2, 0.15mol/L PBS (containing 10% calf serum) in a petri dish, and incubate at room temperature for 15 minutes;
⑤ Wash the petri dish 3 times with pH 7.2, 0.15mol/L PBS, overnight at 4°C, or put it at -20°C for later use. The storage time should not exceed 2 to 3 months.

(2) Negative selection method (enrichment of CD8+ cells)
①Take heparin anticoagulated peripheral blood, separate mononuclear cells with Ficoll-Dipatic meglumine stratification solution, separate T cells with E rosette technique, and use Hank’s solution to prepare T cell pellets to 3～4×106/ml;
②Take the T cell suspension to react with an appropriate amount of anti-CD4 monoclonal antibody, and react for 30 minutes at 4°C;
③ Wash the cells twice with Hank’s solution, and centrifuge at 1000 r/min for 10 minutes each time to remove unbound monoclonal antibodies;
④ Take 3ml of cell suspension and add it to a petri dish coated with goat anti-mouse IgG antibody, and react at room temperature for 1 hour. Pay attention to gently shake once at an interval of 10 to 15 minutes;
⑤ Use 5ml Hank’s solution to gently wash the non-adhered cells, usually 4 times, and store the collected cells (ie CD8+ cells) at 4°C. Note: Avoid direct contact with the moving plate with a straw.

(3) Positive selection method to separate adherent cells (enrichment of CD4+ cells)
① Prepare lidocaine hydrochloride with Hank’s solution, the final concentration is 4mg/ml;
② Add 3 to 4 ml of lidocaine solution to the washed petri dish, let it stand at room temperature for 10 to 15 minutes, and blow the cells with a sharp pipette until all the adhered cells are peeled off;
③ Wash the plate with Hank’s solution, centrifuge the cells at 1000r/min, 10min, and wash the cell pellet with Hank’s solution 3 times to obtain CD4+ cells.
In the same way, if the T cell suspension reacts with the CD8 monoclonal antibody, CD4+ cells can be obtained by the negative selection method, and CD8+ cells can be obtained by the positive selection method.
(4) Store the obtained CD4+ and CD8+ cells at 4°C for later use, and check the purity and viability of the cells.

[Result analysis]
The viability of the cells separated by this method is greater than 98%, and the purity of the separated cells is detected by direct or indirect immunofluorescence.
Positive selection method
CD4+ cells range 89%～100%
CD8+ cells range 85%～100%
Negative selection method
CD4+ cells range 78%～94%
CD8+ cells range 77%～88%

[Precautions]
(1) This technology requires sufficient monoclonal antibodies to be adsorbed on the plastic surface, and the removed cells will not spontaneously adhere to the culture dish.
(2) Adjust the volume of the cell suspension appropriately according to the area of ​​the bottom of the culture dish.
(3) In this experiment, sterile culture flasks can be used instead of petri dishes, because protein-coated culture flasks are easier to store than petri dishes.
(4) In order to minimize non-specific binding caused by adhesion to the solid substrate, Ca2+ and Mg2+-free culture medium is required.
(5) The mouse anti-human T cell monoclonal antibody prepared each time is not consistent, so the dilution factor of the antibody must be determined through preliminary experiments after each preparation.