TITLE: Measuring relative reactivity of mouse TCRs against a mouse cancer cell line
AUTHORS: Bulent Arman Aksoy, Pinar Aksoy, Elinor Gottschalk, Jeff Hammerbacher

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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).</div></div>

[STEPS]
?. [Preparation of electroporation material]
Order, clone, and midi-prep all the TCR and mouse CD8 plasmids:and make sure they are of good quality for further applications.
?. [Preparation of electroporation material]
Linearize plasmids using the corresponding enzymes right at the end of their insertsPreferred enzyme for the OT-I plasmids is EcoRI: and the preferred enzyme for the mouse CD8s is NotI:We recommend the following restriction reaction: of plasmid DNA of the corresponding restriction enzyme of the CutSmart Buffer (10X)Top the reaction with nuclease-free water to Incubate the reaction at  for at least  .
50 µg
25 µl
25 µl
250 µl
37 °C
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.
?. [Preparation of electroporation material]
Extract the linearized DNA via the standard phenol:chloroform extraction protocol:Add  of nuclease-free water so that the final volume for the restriction reaction is Add  of phenol:chloroform and vortex wellSpin at Transfer the (top) aqueous layer to a new tube, add  chloroform, and vortex wellSpin at Transfer the (top) aqueous layer to a new tube, add  absolute EtOH, and mix well by inverting the tube a few timesKeep the sample at  for at least Spin at Discard the supernatant without losing the pellet and add  70% EtOH to wash the pelletSpin at Discard the supernatant, remove all the residual alcohol, and resuspend the pellet in  nuclease-free water
250 µl
500 µl
500 µl
Centrifuge: 14000 33, 5 min, 4 10
500 µl
Centrifuge: 14000 33, 5 min, 4 10
1000 µl
-20 °C
Centrifuge: 14000 33, 30 min, 4 10
500 µl
Centrifuge: 14000 33, 10 min, 4 10
50 µl
?. [Preparation of electroporation material]
In vitro transcribe mRNA using the linearized templates using NEB's mRNA synthesis and LiCl isolation protocols.Store the IVT'ed mRNA at  for future use.
We recommend starting with   of linearized template for each product and scaling the NEB's recommended reaction by 10X. Our preferred final elution volume is  , which should yield 1.5 - 2 ug/uL mRNA.
-80 °C
?. [Culturing and expanding effector and target cells (Day -3)]
Thaw the Jurkat-NFAT cells that come with the T cell bioactivity kit:and culture them by seeding 5 million cells in  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.
50 ml
Jurkat media:   of Corning™ RPMI 1640 Medium (Mod.) 1X with L-Glutamine   of Fetal Plus®   of Penicillin-Streptomycin (10,000 U/mL)
?. [Co-culture setup (Day 0)]
Electroporate Jurkats with mouse CD8 and OT-I subunits
?. [Culturing and expanding effector and target cells (Day -3)]
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.
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)]
Fill 4 wells of a 6-well culture plate with of warm Jurkat media. We will be using this plate as our recovery plate after the electroporation.
6 ml
?. [Co-culture setup (Day 0)]
Spin them down at Re-suspend them in  of PBS (first wash)Spin them down at Re-suspend them in  of PBS (second wash)Re-suspend them in  of R buffer
Centrifuge: 350 34, 5 min, 4 10
25 ml
Centrifuge: 350 34, 5 min, 4 10
25 ml
900 µl
?. [Co-culture setup (Day 0)]
Add of mouse CD8A mRNA (~ ) of mouse CD8B mRNA (~ ) of mouse OT-I alpha mRNA (~ ) of mouse OT-I beta mRNA (~ )onto the  Jurkat cell suspension in R buffer and make sure you mix them well
25 µl
40 µg
25 µl
40 µg
25 µl
40 µg
25 µl
40 µg
900 µl
?. [Co-culture setup (Day 0)]
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.
?. [Co-culture setup (Day 0)]
Let the electroporated Jurkat cells recover for at least 12 hours.
?. [Co-culture setup (Day 0)]
Seed MC38s into two solid-bottom white plates through 2-fold dilutions:
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   of media per well. To have 100K cells in   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.
?. [Co-culture setup (Day 0)]
Let the targets cells attach and settle down for at least 12 hours.
?. [Assay luciferase activity (Day 1)]
Replenish Jurkats with fresh media:Collect and combine all electroporated cells into a single 50-mL falcon tubeSpin   Discard the supernatantRe-suspend in   of fresh Jurkat media (~ 1.3 million cells per mL)Split the cell suspension into two (5 mL each) 15-mL falcon tubesSpin Discard the supernatantRe-suspend in   of fresh Jurkat media (~ 1.3 million cells per mL)Split the cell suspension into two (5 mL each) 15-mL falcon tubesRe-suspend in  of fresh Jurkat media (~ 1.3 million cells per mL)Label and pulse one of the tubes with the SIINFEKL peptide at  (9.63 ug/mL)
Centrifuge: 350 34, 5 min, 4 10
10 ml
?. [Assay luciferase activity (Day 1)]
Aspirate the media from the MC38-seeded plates
?. [Assay luciferase activity (Day 1)]
Add  (~100K) of the electroporated Jurkats onto each well. Label the plates as pulsed or unpulsed accordingly.This should give us the following plate setup:
75 µl
To reduce the chances of cross-contamination, always start by setting up the co-culture for the unpulsed condition.
?. [Assay luciferase activity (Day 1)]
Co-culture for at least .
?. [Assay luciferase activity (Day 1)]
Take the plates and the luciferase substrates out and let them equilibrate at   for  .
0 Room temperature
?. [Assay luciferase activity (Day 1)]
Add  of the luciferase reagent onto each well and let the reactions run for at .
75 µl
?. [Assay luciferase activity (Day 1)]
Measure the luciferase activity using a standard plate reader with luminescence reading capability