Isolation and expansion of human NK T cells

Summary

This protocol allows the identification and quantitative analysis of trace amounts of N K T cells. Effective measures should be taken to block non-specific staining to obtain reliable results. Two monoclonal antibodies were used to ensure the specificity of the results. Author: J.E. Colligan et al, Translator: Xuitao Cao et al, This experiment is from the "Compendium of Immunology Laboratory Guidelines".

Operation method

Isolation and Amplification of Human NKT Cells

Move

Basic Scheme 1 Identification of NK T cells Material

Peripheral heparin anticoagulated blood

P B S

R P M I -1640 medium with 10 % (V / V ) human serum or F C S and 10U /m l I L -2 (optional)

flow cytometry buffer (FCbuffer): PBS with 1 % (v/V) human serum, 1 % (V/V) FBS and 0.1 % (m/V) sodium azide

Fluorescein-labeled anti-Va24 monoclonal antibody (cloneC15B 2, PE or FITC labeled, Coulter-Immunotech)

Fluorescein-labeled anti-Vpil monoclonal antibody (clone C 21D 2, PE or FITC labeled, Coulter)

BDPharmingen-labeled isotype control antibody (BDPharmingen)

Synaptophysin-labeled anti-6B 1 1 monoclonal antibody (B D Pharmingen; optional)

Cy5-labeled anti-C D 4 monoclonal antibody, Cy5-labeled anti-C D 8 monoclonal antibody
(BDPhanningen, optional)

Fluorescein-labeled anti-C D 161 monoclonal antibody (Coulter, recommended)

Fluorescein-labeled other relevant antibodies (optional)

PBS with 4 % paraformaldehyde (PF A): Prepare in a fume hood, heat to 80°C with stirring to accelerate dissolution, dispense cold and store at 20°C.

0.5 ml microcentrifuge tubes or 96-well cell culture plates.

Flow cytometer

1 . Prepare human peripheral heparin anticoagulated blood (5 to 10 ml), which can be stored at room temperature for up to I d, depending on conditions. Ficoll-Hypaque Dense

Option 2: Separation of NK T cells by immunomagnetic beads and subsequent expansion

This method isolates and expands NK T cells (< 10,000 NK T cells) from a few milliliters of peripheral blood. Concentrated leukocytes (a by-product of platelet isolation) can also be used as a source of NK T cells.

Materials (see Appendix 1 for items with V)

Peripheral heparin anticoagulation

P B S with 2m m o l / L E D T A (Appendix 1)

Unlabeled anti-V 〇 t 2 4 monoclonal antibody (Coulter)

Unlabeled anti-6B11 monoclonal antibody, i.e. anti-TCRot monoclonal antibody (B D Pharmingen)

Options Flow Sorting and Expansion of I N K T Cells Additional Materials
Option 2 Cloning of NK T cells Additional materials (see basic options 1 and 2 for additional materials) 1

T-cell cloning medium: RPMI-1640, containing 10 % (WV) autologous human serum, 20 U/mL IL-2 and 20 U/ml IL-7 phytohemagglutinin-P (PHA-P; Difco).

1. Separate TOMC by Ficoll-Hypaque density gradient centrifugation (Unit 8.1). A portion of the cells is removed, inactivated by irradiation and used as feeder cells.

2 . The remaining cells were washed with PBS, resuspended with PBS containing 10 % human serum, adjusted to a concentration of ^IO8 cells/m l, and incubated on ice for 15 min.

3 . Add fluorescein-labeled anti-Va24 monoclonal antibody and another fluorescein-labeled anti-Vpil monoclonal antibody (or labeled anti-6B11 monoclonal antibody, recommended), both at a final concentration of IOiU g An U ice bath for 30 min. At the same time, take a portion of the cells and add the corresponding isotype control antibody.

4 . In a 96-well round-bottom culture plate, add IO5 photoinactivated (5000 rad) autologous feeder cells and PHA-P (final concentration 2ug/ml), and replenish the T-cell cloning medium to a final volume of 0.1m l. At the same time, sort the cells by a flow sorter. At the same time, Va24+ Vpll+ (or 6B11+) double-positive cells should be sorted by flow sorter, and the sorted cells should be added directly into the 96-well plate mentioned above.

5.-After a week, add 0.1 ml of T cell cloning medium (without PHA-P) to each well. Observe cell growth under the microscope: after 10~M d, one or more mother cell clones (mother cells are round and large in size, with more than 100 cells per clone) will appear in the positive wells.

6 . According to the growth of the clones, passaging with T cell cloning medium is performed every 2 to 3 d. If autologous serum is used for T-cell cloning, add 25% allogeneic human serum or FBS to the culture medium at each passaging after the cells have expanded until the autologous serum is completely replaced.

7 . When the number of cells was sufficient, the expression of V a 2 4 and Vpll ( or 6B 1 1 ) in different clones was examined by flow cytometry.

After 3 to 4 weeks, the cells can be given a second stimulation or continue to be cultured in the T cell cloning medium for several weeks.

Secondary Stimulation and Cloning of NK T Cells by Adjuvant Programs Materials

4. On day 1, add 50U/ml each of recombinant IL-2 and IL-7.

5 . On day 5, half-volume exchange with fresh medium was performed to maintain the concentration of each cytokine at 50U /m l .

6 . On day 10, observe the expansion of T cell clones under the microscope. If the mother cell density is high and the medium turns yellow, pass the cells 1:2 with cytokine-containing expansion medium. For vigorously growing cells, passaging can be done every 2 to 3 d.

The cells can be expanded about 1000 times in about 2 weeks. Cells can continue to be cultured for several weeks without additional stimulation.

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