Aug 27, 2024

Public workspaceMitochondrial Antigen Presentation (MitAP) to 2CZ CD8+ T cell hybridoma

DOI
dx.doi.org/10.17504/protocols.io.5qpvok9j7l4o/v1
  • 1McGill Research Centre on Complex Traits and Department of Microbiology and Immunology, McGill University
Alexandra Kazanova
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DOI: dx.doi.org/10.17504/protocols.io.5qpvok9j7l4o/v1
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 07, 2024
Last Modified: August 27, 2024
Protocol Integer ID: 101940
Keywords: ASAPCRN
Funders Acknowledgement:
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:

  • ReagentRecombinant 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
AB
Heat inactivated FBS (Wisent)10%
NEAA1%
Sodium pyruvate1%
Hepes1mM
penicillin/streptomycin1%
  • PBS without Ca2+/Mg2+
  • 1M Glycine in PBS
  • 4% paraformaldehyde (PFA)
  • SIYRYYGL peptide (Genscript) 0.5 mg/ml
  • Tag-it Violet (Biolegend, Cat#455101)
  • ReagentLPS-EB (LPS from E. coli O111:B4)InvivoGenCatalog #tlrl-3pelps

9 days prior to experiment
9 days prior to experiment
Collect bone marrow from femur of mice and start Bone Marrow Dendritic Cell (BMDC) culture with Amount20 undetermined mrGM-CSF (according to BMDC protocol).

Three days prior to experiment (minimum)
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 (Concentration2 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:
On the day of experiment:
14h 40m
14h 40m
Stimulate your antigen presenting cells (APC) with 80-100 ug/ml of Helicobacter pylori and sonicate for Duration06:00:00 . Control - unstimulated APC.

6h
Collect APC gently with cell scraper into 50 ml tubes, wash with ice cold PBS and spin down at Centrifigation600 x g, 4°C, 00:05:00 .

5m
Centrifigation
Wash
Discard supernatant and resuspend APC pellet in Amount2 mL of 1% PFA (dilute stock 4% PFA in PBS), leave for fixation Duration00:09:00 at TemperatureRoom temperature .

9m
Quench PFA with at least 9 times exceeding volume (with Amount18 mL in case of Amount2 mL PFA) of Concentration0.1 Molarity (M) Glycine in complete RPMI (dilute Concentration1 Molarity (M) Glycine with complete RPMI 1:9).

Spin Centrifigation600 x g, 4°C, 00:05:00 and decant.

5m
Centrifigation
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.

Wash
Gently mix a pellet of 2cz cells with Amount1 mL of cell tracker diluted in PBS to Concentration0.63 micromolar (µM) (Tag-it Violet Biolegend).

Mix
Incubate Duration00:12:00 at Temperature37 °C .

12m
Incubation
Add Amount1 mL of FBS and incubate another Duration00:05:00 at TemperatureRoom temperature in the dark.

5m
Incubation
Pipetting
Add Amount13 mL of complete RPMI mix and spin down at Centrifigation400 x g, 4°C, 00:05:00 .

5m
Centrifigation
Pipetting
Wash two more times with complete RPMI, count and bring to a concentration 0.5x10^6 cell/ml.

Wash
Co-culture at Temperature37 °C 5% CO2 DurationOvernight fixed APC with Tag-itViolet+2CZ in round bottom 96 well plate in technical triplicates. 50,000 2cz cells – Amount100 µL and 100,000 APC in another Amount100 µL per well.

  • Negative control Amount100 µL of 2cz cells plus Amount100 µL of complete RPMI; Positive controlAmount100 µL of 2cz cells plus Amount100 µL of Amount0.2 undetermined SIYRYYGL peptide in complete RPMI.

8h
Next morning
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