Aug 27, 2024
Mitochondrial Antigen Presentation (MitAP) to 2CZ CD8+ T cell hybridoma
- 1McGill Research Centre on Complex Traits and Department of Microbiology and Immunology, McGill University
Protocol Citation: Alexandra Kazanova 2024. Mitochondrial Antigen Presentation (MitAP) to 2CZ CD8+ T cell hybridoma. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvok9j7l4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 06, 2024
Last Modified: August 27, 2024
Protocol Integer ID: 101940
Keywords: ASAPCRN, 2cz hybridoma cells in an elispot assay, protocol details the mitochondrial antigen presentation, mitochondrial antigen presentation, producing 2cz hybridoma cell, 2cz cells with flow cytometry, 2cz cell, flow cytometry, assay for the detection, readout for mitap
Funders Acknowledgements:
Aligning Sciences Across Parkinson's
Grant ID: ASAP-000525
Abstract
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.
Materials
Reagents:
- Recombinant Mouse GM-CSF (carrier-free)BioLegendCatalog #576308
- The OGDH/Ld and OGDH/Kb-restricted 2CZ CD8T+ cell hybridoma.
- Complete RPMI media: RPMI 1640 with GLUTAMAX
| A | B | |
| Heat inactivated FBS (Wisent) | 10% | |
| NEAA | 1% | |
| Sodium pyruvate | 1% | |
| Hepes | 1mM | |
| penicillin/streptomycin | 1% |
- PBS without Ca2+/Mg2+
- 1M Glycine in PBS
- 4% paraformaldehyde (PFA)
- SIYRYYGL peptide (Genscript) 0.5 mg/ml
- Tag-it Violet (Biolegend, Cat#455101)
- LPS-EB (LPS from E. coli O111:B4)InvivoGenCatalog #tlrl-3pelps
9 days prior to experiment
Collect bone marrow from femur of mice and start Bone Marrow Dendritic Cell (BMDC) culture with 20 µL mrGM-CSF (according to BMDC protocol).
Three days prior to experiment (minimum)
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).
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).
On the day of experiment:
14h 40m
Stimulate your antigen presenting cells (APC) with 80-100 ug/ml of Helicobacter pylori and sonicate for 06:00:00 . Control - unstimulated APC.
6h
Collect APC gently with cell scraper into 50 ml tubes, wash with ice cold PBS and spin down at 600 x g, 4°C, 00:05:00 .
5m
Discard supernatant and resuspend APC pellet in 2 mL of 1% PFA (dilute stock 4% PFA in PBS), leave for fixation 00:09:00 at Room temperature .
9m
Quench PFA with at least 9 times exceeding volume (with 18 mL in case of 2 mL PFA) of 0.1 Mass Percent Glycine in complete RPMI (dilute 1 Mass Percent Glycine with complete RPMI 1:9).
Spin 600 x g, 4°C, 00:05:00 and decant.
5m
Repeat step 7-8 two more times.
Resuspend in complete RPMI, count cells and bring the concentration of fixed APC to 10ˆ6 cell/ml.
Collect 2CZ cells from a culture flask and wash it in PBS.
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).
Incubate 00:12:00 at 37 °C .
12m
Add 1 mL of FBS and incubate another 00:05:00 at Room temperature in the dark.
5m
Add 13 mL of complete RPMI mix and spin down at 400 x g, 4°C, 00:05:00 .
5m
Wash two more times with complete RPMI, count and bring to a concentration 0.5x10^6 cell/ml.
Co-culture at 37 °C 5% CO2 Overnight 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 µL SIYRYYGL peptide in complete RPMI.
8h
Next morning
Collect cells and proceed to staining for flow cytometry (Viability and activation induced markers (AIM): CD69, CD137; PD1)
For analysis in FlowJo gate on live (Viability stain negative), Tag-it violet+ single cells.
Make a positive gate on CD69/PD1/CD137.
Use Tools → Boolean to create OR gates to acquire total activated 2CZ cells.
Deduct from % AIM+ in samples % of AIM+ in of 2CZ cells cultured without any APC.
Discard results if AIM% of 2CZ with SIYRYYGL well doesn’t exceed 2CZ alone.
Protocol references
Matheoud, D., Sugiura, A., Bellemare-Pelletier, A., Laplante, A., Rondeau, C., Chemali, M., Fazel, A., Bergeron, J. J., Trudeau, L.-E., Burelle, Y., Gagnon, E., McBride, H. M., & Desjardins, M. (2016). Parkinson’s Disease-Related Proteins PINK1 and Parkin Repress Mitochondrial Antigen Presentation. Cell, 166(2), 314–327. doi:10.1016/j.cell.2016.05.039