Jan 14, 2020
Measuring relative reactivity of mouse TCRs against a mouse cancer cell line
- 1Medical University of South Carolina
- Hammer LabTech. support phone: +18437924527 email: arman@hammerlab.org
Protocol Citation: Bulent Arman Aksoy, Pinar Aksoy, Elinor Gottschalk, Jeff Hammerbacher 2020. Measuring relative reactivity of mouse TCRs against a mouse cancer cell line. protocols.io https://dx.doi.org/10.17504/protocols.io.ba8gihtw
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: January 10, 2020
Last Modified: January 14, 2020
Protocol Integer ID: 31720
Keywords: TCR, reactivity, bioactivity, T cell, cytotoxicity, OT-I, mouse, MC38B
Abstract
This protocol repurposes Promega's T Cell Activiation Bioassay workflow to be able to test relative mouse TCR reactivity against a cell line. This specific protocol uses MC38 as the target as it doesn't normally present SIINFEKL and have good H2Kb and H2Db expression levels. The reactivity will be in relative to the positive control (OT-I reactivity against SIINFEKL-pulsed cells) and the negative control (OT-I reactiviy against unpulsed cells).
Materials
MATERIALS
EcoRI-HF - 10,000 unitsNew England BiolabsCatalog #R3101S
NotI-HF - 2,500 unitsNew England BiolabsCatalog #R3189L
HiScribe T7 ARCA mRNA Kit (with Tailing) - 20 rxnsNew England BiolabsCatalog #E2060S
UltraPure™ Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)Thermo Fisher ScientificCatalog #15593031
Nuclease-Free Water
Ethanol (100%, Molecular Biology Grade)Fisher ScientificCatalog #BP2818500
T Cell Activation Bioassay (NFAT)PromegaCatalog #J1621
SpectraMax i3 Multi-Mode Microplate Detection PlatformMolecular DevicesCatalog #i3x
Corning™ RPMI 1640 Medium (Mod.) 1X with L-GlutamineFisher ScientificCatalog #MT10041CV
Fetal Plus®Atlas BiologicalsCatalog # FP-0500-A
Penicillin-Streptomycin (10,000 U/mL)Thermo Fisher ScientificCatalog #15140122
Neon™ Transfection SystemThermo Fisher ScientificCatalog #MPK5000
ChloroformFisher ScientificCatalog #C298-4
Neon™ Transfection System 100 µL KitThermo FisherCatalog #MPK10096
pcDNA3.1( )-OTI-TCRAaddgeneCatalog #131035
pcDNA3.1( )-OTI-TCRBaddgeneCatalog #131036
Cd8a (NM_001081110) Mouse Tagged ORF CloneOriGeneCatalog #MR227539
Cd8b1 (NM_009858) Mouse Tagged ORF CloneOriGeneCatalog #MR225204
CELL CULTURE MICROPLATE 96 WELL PS F-BOTTOM (CHIMNEY WELL) WHITE CELLSTAR® TC LID WITH CONDENSgreiner bio-oneCatalog #655083
STEP MATERIALS
EcoRI-HF - 10,000 unitsNew England BiolabsCatalog #R3101S
NotI-HF - 2,500 unitsNew England BiolabsCatalog #R3189L
pcDNA3.1( )-OTI-TCRAaddgeneCatalog #131035
pcDNA3.1( )-OTI-TCRBaddgeneCatalog #131036
Cd8a (NM_001081110) Mouse Tagged ORF CloneOriGeneCatalog #MR227539
Cd8b1 (NM_009858) Mouse Tagged ORF CloneOriGeneCatalog #MR225204
T Cell Activation Bioassay (NFAT)PromegaCatalog #J1621
SpectraMax i3 Multi-Mode Microplate Detection PlatformMolecular DevicesCatalog #i3x
Protocol materials
Neon™ Transfection SystemThermo Fisher ScientificCatalog #MPK5000
Cd8b1 (NM_009858) Mouse Tagged ORF CloneOriGeneCatalog #MR225204
UltraPure™ Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)Thermo Fisher ScientificCatalog #15593031
Neon™ Transfection System 100 µL KitThermo FisherCatalog #MPK10096
Fetal Plus®Atlas BiologicalsCatalog # FP-0500-A
SpectraMax i3 Multi-Mode Microplate Detection PlatformMolecular DevicesCatalog #i3x
Ethanol (100%, Molecular Biology Grade)Fisher ScientificCatalog #BP2818500
EcoRI-HF - 10,000 unitsNew England BiolabsCatalog #R3101S
ChloroformFisher ScientificCatalog #C298-4
Cd8a (NM_001081110) Mouse Tagged ORF CloneOriGeneCatalog #MR227539
Penicillin-Streptomycin (10,000 U/mL)Thermo Fisher ScientificCatalog #15140122
Cd8b1 (NM_009858) Mouse Tagged ORF CloneOriGeneCatalog #MR225204
NotI-HF - 2,500 unitsNew England BiolabsCatalog #R3189L
pcDNA3.1( )-OTI-TCRAaddgeneCatalog #131035
T Cell Activation Bioassay (NFAT)PromegaCatalog #J1621
Corning™ RPMI 1640 Medium (Mod.) 1X with L-GlutamineFisher ScientificCatalog #MT10041CV
Nuclease-Free Water
SpectraMax i3 Multi-Mode Microplate Detection PlatformMolecular DevicesCatalog #i3x
NotI-HF - 2,500 unitsNew England BiolabsCatalog #R3189L
pcDNA3.1( )-OTI-TCRAaddgeneCatalog #131035
pcDNA3.1( )-OTI-TCRBaddgeneCatalog #131036
EcoRI-HF - 10,000 unitsNew England BiolabsCatalog #R3101S
pcDNA3.1( )-OTI-TCRBaddgeneCatalog #131036
T Cell Activation Bioassay (NFAT)PromegaCatalog #J1621
CELL CULTURE MICROPLATE 96 WELL PS F-BOTTOM (CHIMNEY WELL) WHITE CELLSTAR® TC LID WITH CONDENSgreiner bio-oneCatalog #655083
HiScribe T7 ARCA mRNA Kit (with Tailing) - 20 rxnsNew England BiolabsCatalog #E2060S
Cd8a (NM_001081110) Mouse Tagged ORF CloneOriGeneCatalog #MR227539
pcDNA3.1( )-OTI-TCRAaddgeneCatalog #131035
pcDNA3.1( )-OTI-TCRBaddgeneCatalog #131036
Cd8a (NM_001081110) Mouse Tagged ORF CloneOriGeneCatalog #MR227539
Cd8b1 (NM_009858) Mouse Tagged ORF CloneOriGeneCatalog #MR225204
NotI-HF - 2,500 unitsNew England BiolabsCatalog #R3189L
EcoRI-HF - 10,000 unitsNew England BiolabsCatalog #R3101S
T Cell Activation Bioassay (NFAT)PromegaCatalog #J1621
SpectraMax i3 Multi-Mode Microplate Detection PlatformMolecular DevicesCatalog #i3x
Before start
Make sure you have some familiarity with
- Plasmid propagation, midi-prepping, restriction, and purification
- In vitro transcription and RNA handling
- Electroporation
- Basic cell cell culture maintenance
Preparation of electroporation material
Preparation of electroporation material
Order, clone, and midi-prep all the TCR and mouse CD8 plasmids:
pcDNA3.1( )-OTI-TCRAaddgeneCatalog #131035
pcDNA3.1( )-OTI-TCRBaddgeneCatalog #131036
Cd8a (NM_001081110) Mouse Tagged ORF CloneOriGeneCatalog #MR227539
Cd8b1 (NM_009858) Mouse Tagged ORF CloneOriGeneCatalog #MR225204
and make sure they are of good quality for further applications.
Linearize plasmids using the corresponding enzymes right at the end of their inserts
Preferred enzyme for the OT-I plasmids is EcoRI:
EcoRI-HF - 10,000 unitsNew England BiolabsCatalog #R3101S
and the preferred enzyme for the mouse CD8s is NotI:
NotI-HF - 2,500 unitsNew England BiolabsCatalog #R3189L
We recommend the following restriction reaction:
- 50 µg of plasmid DNA
- 25 µL of the corresponding restriction enzyme
- 25 µL of the CutSmart Buffer (10X)
- Top the reaction with nuclease-free water to 250 µL
Incubate the reaction at 37 °C for at least 01:00:00 .
Note
It is very important to fully linearize the plasmid to prevent potential off-running mRNAs. Based on the incubation time, the amount of enzyme can be reduced but the linearization should always be quality-checked via running the product on agarose gel when in doubt.
Extract the linearized DNA via the standard phenol:chloroform extraction protocol:
- Add 250 µL of nuclease-free water so that the final volume for the restriction reaction is 500 µL
- Add 500 µL of phenol:chloroform and vortex well
- Spin at 14000 rpm, 4°C, 00:05:00
- Transfer the (top) aqueous layer to a new tube, add 500 µL chloroform, and vortex well
- Spin at 14000 rpm, 4°C, 00:05:00
- Transfer the (top) aqueous layer to a new tube, add 1000 µL absolute EtOH, and mix well by inverting the tube a few times
- Keep the sample at -20 °C for at least 00:30:00
- Spin at 14000 rpm, 4°C, 00:30:00
- Discard the supernatant without losing the pellet and add 500 µL 70% EtOH to wash the pellet
- Spin at 14000 rpm, 4°C, 00:10:00
- Discard the supernatant, remove all the residual alcohol, and resuspend the pellet in 50 µL nuclease-free water
- Quality check the final DNA solution and estimate the concentration via Nanodrop.
In vitro transcribe mRNA using the linearized templates using NEB's mRNA synthesis and LiCl isolation protocols.
Note
We recommend starting with 10 µg of linearized template for each product and scaling the NEB's recommended reaction by 10X. Our preferred final elution volume is 250 µL , which should yield 1.5 - 2 ug/uL mRNA.
Store the IVT'ed mRNA at -80 °C for future use.
Culturing and expanding effector and target cells (Day -3)
Culturing and expanding effector and target cells (Day -3)
Thaw the Jurkat-NFAT cells that come with the T cell bioactivity kit:
T Cell Activation Bioassay (NFAT)PromegaCatalog #J1621
and culture them by seeding 5 million cells in 50 mL of Jurkat media within a T75 flask for at least 3 days or until they reach a density of 1.5 million cells per mL.
Note
Jurkat media:
- 500 mL of Corning™ RPMI 1640 Medium (Mod.) 1X with L-Glutamine
- 50 mL of Fetal Plus®
- 5 mL of Penicillin-Streptomycin (10,000 U/mL)
Thaw and start culturing MC38 cells. Seeding ~2 million cells in a T75 flask and culturing them for at least three days should yield enough cells for the co-culture.
Note
We have been using MC38 cells as our effectors but the choice of cell line is up to the experimenter. Ideally, the cell line doesn't present the SIINFEKL peptide on its own (without pulsing) so that we can use it as a negative control when co-cultured with the OT-I TCR. This protocol assumes, the cell line is of adherent nature so any suspension cell line could require some customization.
Co-culture setup (Day 0)
Co-culture setup (Day 0)
Electroporate Jurkats with mouse CD8 and OT-I subunits
Fill 4 wells of a 6-well culture plate with 6 mL of warm Jurkat media. We will be using this plate as our recovery plate after the electroporation.
- Collect 20 million Jurkats
- Spin them down at 350 x g, 4°C, 00:05:00
- Re-suspend them in 25 mL of PBS (first wash)
- Spin them down at 350 x g, 4°C, 00:05:00
- Re-suspend them in 25 mL of PBS (second wash)
- Re-suspend them in 900 µL of R buffer
Add
- 25 µL of mouse CD8A mRNA (~ 40 µg )
- 25 µL of mouse CD8B mRNA (~ 40 µg )
- 25 µL of mouse OT-I alpha mRNA (~ 40 µg )
- 25 µL of mouse OT-I beta mRNA (~ 40 µg )
onto the 900 µL Jurkat cell suspension in R buffer and make sure you mix them well
Using Neon's the 100 uL tips, electroporate cells at 1350 V 10 ms 3 pulse setting. Use one tip for one reaction. Change the E2 solution every 6 electroporation reactions. Recover at most two reactions within each 6-well-plate well. Electroporate at least 8 reactions, which should yield 16 million Jurkat cells.
Equipment
new equipment
NAME
Thermo Fisher Scientific Neon™ Transfection System
BRAND
MPK5000S
SKU
Let the electroporated Jurkat cells recover for at least 12 hours.
Seed MC38s into two solid-bottom white plates through 2-fold dilutions:
Plate set up for the target cells. Have at most 6 replicates (row-wise) and prepare 2-fold serial dilutions for each replicate. We will assume that the cells will replicate once overnight so the numbers will double on the day of the co-culture.
Note
Although the final volume doesn't matter that much since we will be aspirating the media before setting the co-culture, when in doubt you can go with 60 µL of media per well. To have 100K cells in 60 µL of media, the serial dilution should start roughly at 3.3 million cells per mL concentration and we will be needing roughly 1.5 million cells per plate.
Let the targets cells attach and settle down for at least 12 hours.
Assay luciferase activity (Day 1)
Assay luciferase activity (Day 1)
Replenish Jurkats with fresh media:
- Collect and combine all electroporated cells into a single 50-mL falcon tube
- Spin 350 x g, 4°C, 00:05:00
- Discard the supernatant
- Re-suspend in 10 mL of fresh Jurkat media (~ 1.3 million cells per mL)
- Split the cell suspension into two (5 mL each) 15-mL falcon tubes
- Label and pulse one of the tubes with the SIINFEKL peptide at 10 micromolar (µM) (9.63 ug/mL)
Aspirate the media from the MC38-seeded plates
Add 75 µL (~100K) of the electroporated Jurkats onto each well. Label the plates as pulsed or unpulsed accordingly.
Note
To reduce the chances of cross-contamination, always start by setting up the co-culture for the unpulsed condition.
This should give us the following plate setup:
Plate setup for the coculture condition. Jurkat concentration is kept fixed but the target cells are titrated down from 2:1 target:effector ratio using 2-fold serial dilution.
Co-culture for at least 06:00:00 .
Take the plates and the luciferase substrates out and let them equilibrate at Room temperature for
00:10:00 .
Add 75 µL of the luciferase reagent onto each well and let the reactions run for at 00:10:00 .
Measure the luciferase activity using a standard plate reader with luminescence reading capability
SpectraMax i3 Multi-Mode Microplate Detection PlatformMolecular DevicesCatalog #i3x
Dataset
OT-I TCR reactivity against SIINFEKL-pulsed or -unpulsed MC38s
NAME
Expected result
Median luminescence at different target:effector ratios across pulsed (positive) and unpulsed (negative) control samples. Each TCR that will be tested will produce a reactivity metric relative to these two controls.