Aug 23, 2022

Public workspaceMouse lung MAIT cell expansion and purification protocol

DOI
dx.doi.org/10.17504/protocols.io.x54v9yrpqg3e/v1
  • 1University of California, San Diego;
  • 2La Jolla Institute for Immunology
Gabriel A Ascui
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DOI: dx.doi.org/10.17504/protocols.io.x54v9yrpqg3e/v1
External link: https://www.lji.org/labs/kronenberg
Protocol Citation: Gabriel A Ascui, Eleni Phung, Alba Mendis, Mitchell Kronenberg 2022. Mouse lung MAIT cell expansion and purification protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9yrpqg3e/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: March 30, 2022
Last Modified: June 05, 2023
Protocol Integer ID: 60070
Keywords: MAIT cells, in vitro, lung, mouse
Funders Acknowledgement:
NIH
Grant ID: 5R01AI137230-04
Disclaimer
The Authors declare no competing interests.
Abstract
Mucosal Associated Invariant T (MAIT) cells are unconventional T cells present abundantly in human tissues. MAIT cells interact with antigens presented by MR1, an MHC class I protein, to fight microbial infections. They also reportedly activate through cytokines to combat viruses. While MAIT cells exhibit the innate-like characteristic of rapid responses to infections, they also display adaptive-like qualities such as an effector-memory phenotype. Defining the role of MAIT cells in the immune system is essential to understand lung immune responses against bacterial infections. The following protocol outlines the expansion of MAIT cells via a cultured Salmonella Typhimurium BRD509 vaccine strain. Six days after infecting mice retro-pharyngeally with BRD509, MAIT cells were extracted from the lungs. The cells were then enriched and labeled with fluorescence for sorting. Once sorted, MAIT cells were plated and activated with anti-CD3/CD28. The cells were then analyzed with flow cytometry.
Protocol materials
ReagentRed Blood Cell Lysis Buffer Hybri-MaxMerck MilliporeSigma (Sigma-Aldrich)Catalog #R7757
Step 2.1
ReagentCell strainer 70um filterFalconCatalog #352350
Step 2.1
ReagentgentleMACS C-Tube Miltenyi BiotecCatalog #130-093-334
Step 2.1
ReagentSpleen Dissociation Medium 10 x 4 mL STEMCELL Technologies Inc.Catalog #7915
Step 2.1
ReagentLIVE/DEAD™ Fixable Yellow Dead Cell Stain Kit, for 405 nm excitationThermo FisherCatalog #L34967
In 2 steps
ReagentEasySep™ Mouse Streptavidin RapidSpheres™ Isolation Kit For processing 1 x 10^9 cells STEMCELL Technologies Inc.Catalog #19860
Step 2.2
ReagentThe Big Easy EasySep™ Magnet For isolating 1 x 10^9 STEMCELL Technologies Inc.Catalog #18001
Step 2.2
MAIT cell in vivo expansion with BRD509
MAIT cell in vivo expansion with BRD509
Salmonella enterica serotype Typhimurium BRD509 vaccine strain culture

Safety information
Work in BSL2 biosafety conditions.

6d
Day 0: Overnight culture of Salmonella Typhimurium BRD509 strain.

  1. Prepare Amount5 mL of LB media with Concentration100 µg/ml of Streptomycin.
  2. Pick Temperature-80 °C BRD509 stock and inoculate tube.
  3. Incubate DurationOvernight in agitationShaker225 rpm, 37°C .


10m
Day 1: Prepare 4h culture of BRD509.

  1. Take Amount1 mL of overnight BRD509 culture and dilute with LB media until Amount5 mL .
  2. Culture for an additional Duration04:00:00 to Duration06:00:00 .
  3. Measure OD600 on spectrophotometer.
  4. Calculate the amount of culture that will need to be diluted in Amount900 µL of sterile PBS to get 10^6 CFU per mouse based on the following conversion table (input OD600):


ABCDEF
A6001A600 of culture0.6
CFU/mL600000000CFU/mL360000000
Desired CFU/mouse2.7777777777777777uL per mouse
Resuspend in 900uL (30uL per mouse)83.33333333333333uL for 30 mice
A600 BRD509 spreadsheet

5h
Day 1: Infection Mice

1. Anesthetize the mice with isoflurane gas.
2. Administer retro-pharyngeal injections of Amount30 µL of diluted BRD509 per mouse.
3. Prepare dilutions of the original dose at 10^4 and 10^5 in sterile PBS.
4. Plate dilutions on LB agar plate with Streptomycin. Count colonies next day to calculate actual dose.
1h
MAIT cell sorting
MAIT cell sorting
2h
2h
Prepare Lungs MAIT cells
Safety information
Work in BSL2 biosafety conditions.

Day 6: Collect lungs

1. Collect lungs from infected mice and process inReagentgentleMACS C-Tube Miltenyi BiotecCatalog #130-093-334wwith Amount2 mL of ReagentSpleen Dissociation Medium 10 x 4 mL Stemcell TechnologiesCatalog #7915 using program 37C_m_LDK_1. This should take about Duration00:30:00 .
2. Filter single cell suspension through ReagentCell strainer 70um filterFalconCatalog #352350 on 50 mL conical tube. Mash any remaining bits of tissue through the strainer with a syringe plunger. Wash strainer with HBSS with 10% FBS until Amount25 mL .
3. Centrifuge at Centrifigation1500 rpm, 4°C, 00:05:00 .
4. Lyse red blood cells by adding Amount1 mL of ReagentRed Blood Cell Lysis Buffer Hybri-MaxSigma AldrichCatalog #R7757 . Incubate at TemperatureRoom temperature for Duration00:05:00 .
5. Add Amount14 mL of HBSS 10%FBS and centrifuge at Centrifigation1500 rpm, 4°C, 00:05:00 .
6. Re-suspend cells in Amount2 mL of MACS buffer.
45m
MAIT cell enrichment

  1. Add 50 ul of Rat Serum to sample.
  2. Add the following biotin-conjugated antibodies:
ABC
Antigen Stock [ ] (mg/ml) V in 2 ml (ul)
CD11b 0.5 4
CD11c 0.5 4
Ter119 0.5 4
F4/80 0.5 4
B220 0.5 4
Ly6G 0.5 4
Biotin-conjugated antibodies
3. Incubate atTemperatureRoom temperature for Duration00:10:00 .
4. Vortex ReagentEasySep™ Mouse Streptavidin RapidSpheres™ Isolation Kit For processing 1 x 10^9 cells Stemcell TechnologiesCatalog #19860 for Duration00:00:30 . Add Amount50 µL of RapidSpheres to sample.
5. Mix and incubate for Duration00:05:00 at TemperatureRoom temperature .
6. Add Amount2 mL MACS Buffer.
7. Place tube into ReagentThe Big Easy EasySep™ Magnet For isolating 1 x 10^9 Stemcell TechnologiesCatalog #18001 . Incubate for Duration00:05:00 at TemperatureRoom temperature .
8. Gently pour supernatant in a new 5 ml tube.
20m 30s
Fluorescent labelling

  1. Stain cells with PE-conjugated or mouse MR1 tetramer loaded with 5-OP-RU at 1:300 dilution for Duration00:45:00 at TemperatureRoom temperature . Wash with PBS 2% FBS and centrifuge at Centrifigation1500 rpm, 4°C, 00:05:00 .


Note
Mouse MR1 tetramers were provided by the NIH Tetramer Core Facility:
https://tetramer.yerkes.emory.edu/


  1. Stain cells with ReagentLIVE/DEAD™ Fixable Yellow Dead Cell Stain Kit, for 405 nm excitationThermo FisherCatalog #L34967 at 1:500 dilution, FcBlock (2G4) at 1:500 dilution and 1:500 dilution of Concentration1 mg/mL Free Streptavidin in PBS.
Note
NOTE: Make sure the PBS has no other protein content.

3. Incubate at Temperature4 °C for Duration00:15:00 . Wash with PBS 2% FBS.
4. Stain cells with the following antibodies:

ABCDEFG
# Marker Population Channel Host Clone Dilution
2 gd TCR DUMP PerCP-Cy5.5 Mouse GL3 1:400
3 IgD DUMP PerCP-Cy5.5 Rat 11-26c.2a 1:300
4 CD11b DUMP PerCP-Cy5.5 Rat M1/70 1:300
5 CD11c DUMP PerCP-Cy5.5 Rat N418 1:300
6 B220 DUMP PerCP-Cy5.5 Rat RA36B2 1:300
7 5-OP-RU MAIT cells PE - - 1:300
8 TCR beta T cells APC-eF780 Mouse H57-597 1:300
9 Live/Dead Yellow BV570 Rat MEL14 1:200
10 CD45 Lymphocytes BV785 Rat 30.F11 1:300
MAIT cell Sorting Panel

4. Incubate atTemperature4 °C for Duration00:30:00 .
5. Wash cells with PBS 2% FBS. Resuspend in Amount4 mL .
1h 35m
Cell Sorting

  1. Sort MAIT cells with 85 nm nozzle on low pressure. Sort into Amount500 µL of sterile FBS in 5 ml FACS tubes.
  2. Gate samples as following: Lymphocytes/Singlets/Live/DUMP-CD45+/Tetramer+TCRbint
Expected result
You should obtain a 20-30% frequency of MAIT cells from the DUMP-CD45+ gate. Total number of MAIT cells should be between 20,000-50,000 per lung.


In vitro culture
In vitro culture
2h
2h

Day 1: MAIT cell activation with anti-CD3/CD28
Coat 48-well plate

While MAIT cells are been sorted, prepare anti-CD3e + anti-CD28 coated plates.

1. Dilute anti-CD3e (2C11) to Concentration5 µg/ml and anti-CD28 to Concentration2 µg/ml in sterile PBS.
2. Add Amount100 µL to each well of a 48-well plate.
3. Incubate at Temperature37 °C for Duration02:00:00 .
4. Wash plate with Amount200 µL of PBS, twice.
5. remove all liquid from every well.

2h
Plate MAIT cells


Note
  • MAIT cell culture media: RPMI 10% FBS 1X Pen/Strep 1X Pyruvate 1X HEPES 55 uM β-mercaptoethanol.


1. Wash sorted cells with MAIT cell culture media.
2. Resuspend cells in MAIT cell at 0.5 x 10^6 cells/mL in MAIT cell culture media supplemented with the following cytokines:
ABCD
Cytokine Final [   ] Stock [   ] Dilution
IL-2 10 ng/ml 20 ug/ml 1:2000
IL-7 20 ng/ml 20 ug/ml 1:1000
IL-1b 10 ng/ml 100 ug/ml 1:1000
IL-23 10 ng/ml 10 ug/ml 1:1000
Cytokines

3. Plate 500,000 cells per well.
10m
Day 2: Put in fresh plate.
5m
Analyze MAIT cells through flow

  1. Stain cells with PE-conjugated or mouse MR1 tetramer loaded with 5-OP-RU at 1:300 dilution for Duration00:45:00 at TemperatureRoom temperature . Wash with PBS 2% FBS and centrifuge at Centrifigation1500 rpm, 4°C, 00:05:00 .
  2. Stain cells with ReagentLIVE/DEAD™ Fixable Yellow Dead Cell Stain Kit, for 405 nm excitationThermo FisherCatalog #L34967 at 1:500 dilution, FcBlock (2G4) at 1:500 dilution and 1:500 dilution of Concentration1 mg/mL Free Streptavidin in PBS.
Note
NOTE: Make sure the PBS has no other protein content.

3. Incubate at Temperature4 °C for Duration00:15:00 . Wash with PBS 2% FBS.
4. Stain cells with the following antibodies:

ABCDEFGHIJ
# Marker Population Channel Host Clone Dilution
2 gd TCR DUMP PerCP-Cy5.5 Mouse GL3 1:400
3 IgD DUMP PerCP-Cy5.5 Rat 11-26c.2a 1:300
4 CD11b DUMP PerCP-Cy5.5 Rat M1/70 1:300
5 CD11c DUMP PerCP-Cy5.5 Rat N418 1:300
6 B220 DUMP PerCP-Cy5.5 Rat RA36B2 1:300
7 5-OP-RU MAIT cells PE - - 1:300
8 TCR beta T cells APC-eF780 Mouse H57-597 1:300
9 Live/Dead Yellow BV570 Rat MEL14 1:200
10 CD45 Lymphocytes BV785 Rat 30.F11 1:300

4. Incubate atTemperature4 °C for Duration00:30:00 .
5. Wash cells with PBS 2% FBS. Resuspend in Amount4 mL .
6. Analyze cells through flow.

1h 35m