NK stimulation assay

NK stimulation assay

>A. Buffers and media

1.  R10

            RPMI 1640 containing:

            10% FBS

            100 units/ml Penicillin/100mg/ml Streptomycin

            55 μM 2-Mercaptaethanol

            2mM L-glutamin

2.  PBA (FACS staining buffer)

            PBS

            0.5% (w/v) BSA

            0.02% Sodium azide

3. RBC Lysis buffer

>B. Harvest (responder) splenocytes

1.  Harvest spleens from B6 mice under sterile conditions (1 mouse yields 70-100x10⁶ splenocytes).

2.  Crush spleen with a plunger of a 3 ml syringe through a 70 μm cell strainer (BD Falcon #352350) in a 6 wells plate containing 3 ml R10.

3.  Wash the strainer and well with R10 and transfer the cells to a 15 ml conical.

4.  Spin down 6 min 1700rpm @ 4°C.

5.  Resuspend the cell pellet in 1 ml RBC lysis buffer.

6.  Incubate 5 min at room temperature (RT)

7.  Add 10 ml R10 and filter trough a 50 μm cell strainer (Partec #04-004-2327).

8.  Take a sample for counting and spin down the remainder 5 min 1600rpm @ 4°C.

9.  Resuspend the cell pellet in R10 at 12-20x10⁶ cells/ml.

>C. Harvest stimulator cells (CHO cells)

1.  Harvest desired (CHO) cell lines. Briefly, discard medium, wash the cells with PBS and add Trypsin/EDTA (3ml for a T185 is sufficient). Put the cells in the incubator for approximately 3-5 minutes (longer incubation times may cleave proteins from the cell surface).

2.  Add 10 ml R10 and transfer the cells to a 15ml conical.

3.  Take a sample for counting and spin down the remainder 5 min 1600rpm @ 4°C.

4.  Resuspend the cell pellet in R10 at 0.67x10⁶ cells/ml.

>D. Setup stimulation assay

1.  Prepare Qdm peptide to 300 μM in R10

2.  Prepare 10X Monensin/CD107a mix: dilute CD107a eFluor660 (1:100) and 1000X Monensin (1:100) in R10

3.  Pipette reactions together in a 24-wells plate:

            Responder splenocytes (3-5x10⁶)                   250 μl                                      

            Stimulator CHO cells (1x10⁵)                        150 μl                                      

            Qdm peptide                                                   50 μl                                        

            Monensin/CD107a mix (add after 1 hour)   50 μl                                        

            Total                                                                500 μl                                      

4.  After mixing, incubate reactions in a 37°C 5%CO₂ incubator.

5.  After total 6-8 hours incubation add 1ml PBA to each well and proceed to FACS staining.

>E. FACS staining

1.  Spin down the cells and remove supernatant 

3.  Add 200 ul PBA and transfer the cell-suspensions to a V-bottom plate 

4.  Spin down 4 min 1600rpm @ 4°C, discard supernatant.

5. Add 50 ul viability stain in PBS

6. Incubate 30 min on ice in the dark.

7. Add 150 ul PBA and spin down 4 min 1600rpm @ 4°C, discard supernatant.     

8. Add 25 ul cell surface antibody mix in Fc block (2.4g2) supernatant.

9. Incubate 30 min on ice in the dark.

10. Add 150 ul PBA and spin down 4 min 1600rpm @ 4°C, discard supernatant.

11. Add 50ul Fix/Perm buffer

12. Incubate 20 min on ice in the dark.

13. Add 150 ul Perm/Wash buffer and spin down 4 min 1600rpm @ 4°C, discard supernatant.

14. Add 150 ul Perm/Wash buffer and spin down 4 min 1600rpm @ 4°C, discard supernatant.

15. Add 25 ul Perm/Wash buffer containing ani-IFNγ antibody.

16. Incubate 30 min on ice in the dark.

17. Add 150 ul Perm/Wash buffer and spin down 4 min 1600rpm @ 4°C, discard supernatant.

18. Take pellet up in 200 μl PBA and filter trough mesh.

19. Analyze by flow cytometry, acquire 5x10⁵ events which should yield approximately 5,000 NK cells. We typically define NK cells as viable CD3-CD19-NKp46+NK1.1+.

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