TITLE: Mitochondrial Antigen Presentation (MitAP) to 2CZ CD8+ T cell hybridoma
AUTHORS: Alexandra Kazanova

[DESCRIPTION]
This protocol details the Mitochondrial Antigen Presentation (MitAP) to 2CZ CD8+ T cell hybridoma. Previously, quantification of the number of IL-2 producing 2cz hybridoma cells in an ELISPOT assay was used as a readout for MitAP (Matheoud et al., 2016). Here, we adapted the assay for the detection of activation-induced markers (AIM) on 2cz cells with flow cytometry.

[STEPS]
SECTION: 9 days prior to experiment
1. Collect bone marrow from femur of mice and start Bone Marrow Dendritic Cell (BMDC) culture with 20 1585 mrGM-CSF (according to BMDC protocol).

SECTION: Three days prior to experiment (minimum)
2. Start 2CZ hybridoma from a frozen stock. Maintain the hybridomas in RPMI-1640 medium supplemented with 5% (v/v) FCS, glutamine (2 millimolar (mM), penicillin (100 U ml−1) and streptomycin (100 μg ml−1).

SECTION: Three days prior to experiment (minimum)
3. Culture 2CZ cells at no point exceeding 10ˆ6 cell/ml in complete RPMI in flasks for suspension cell culture (2CZ on average undergo 2 divisions per day).

SECTION: On the day of experiment:
4. Stimulate your antigen presenting cells (APC) with 80-100 ug/ml of Helicobacter pylori and sonicate for 360 min. Control - unstimulated APC.

SECTION: On the day of experiment:
5. Collect APC gently with cell scraper into 50 ml tubes, wash with ice cold PBS and spin down at 600 x g, 5 min, 4 °C.

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6. Discard supernatant and resuspend APC pellet in 2 mL of 1% PFA (dilute stock 4% PFA in PBS), leave for fixation 9 min at Room temperature.

SECTION: On the day of experiment:
7. Quench PFA with at least 9 times exceeding volume (with 18 mL in case of 2 mL PFA) of 0.1 Molarity (M)Glycine in complete RPMI (dilute 1 Molarity (M) Glycine with complete RPMI 1:9).

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8. Spin 600 x g, 5 min, 4 °C and decant.

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9. Repeat step 7-8 two more times.

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10. Resuspend in complete RPMI, count cells and bring the concentration of fixed APC to 10ˆ6 cell/ml.
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11. Collect 2CZ cells from a culture flask and wash it in PBS. 

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12. Gently mix a pellet of 2cz cells with 1 mL of cell tracker diluted in PBS to 0.63 micromolar (µM)(Tag-it Violet Biolegend).

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13. Incubate 12 min at  37 °C .

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14. Add 1 mLof FBS and incubate another 5 min at  Room temperature  in the dark.

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15. Add 13 mL of complete RPMI mix and spin down at 400 x g, 5 min, 4 °C .

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16. Wash two more times with complete RPMI, count and bring to a concentration 0.5x10^6 cell/ml.

SECTION: On the day of experiment:
17. Co-culture at 37 °C 5% CO2 5 min fixed APC with Tag-itViolet+2CZ in round bottom 96 well plate in technical triplicates. 50,000 2cz cells – 100 µL and 100,000 APC in another 100 µL per well.

Negative control 100 µL of 2cz cells plus 100 µL of complete RPMI; Positive control100 µL  of 2cz cells plus 100 µL of 0.2 1527 SIYRYYGL peptide in complete RPMI.

SECTION: Next morning
18. Collect cells and proceed to staining for flow cytometry (Viability and activation induced markers (AIM): CD69, CD137; PD1)

SECTION: Next morning
19. For analysis in FlowJo gate on live (Viability stain negative), Tag-it violet+ single cells.

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20. Make a positive gate on CD69/PD1/CD137.

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21. Use Tools → Boolean to create OR gates to acquire total activated 2CZ cells.

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22. Deduct from % AIM+ in samples % of AIM+ in of 2CZ cells cultured without any APC.

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23. Discard results if AIM% of 2CZ with SIYRYYGL well doesn’t exceed 2CZ alone.