Jul 31, 2023
TCR sequencing and activation
- 1Shraga Segal Department of Microbiology, Immunology, and Genetics, Ben-Gurion University of the Negev, 8410501, Beer-Sheva, Israel
Protocol Citation: Zorea Jonathan 2023. TCR sequencing and activation. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkokpxv5r/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: July 30, 2023
Last Modified: July 31, 2023
Protocol Integer ID: 85701
Abstract
This protocol is a procedure we follow to isolate tumor infiltrating T cells and try to activate them with unique peptides and to characterize their TCR repertoire
Tumor implantation
Tumor implantation
Grow sufficient amount of cells (5*10^6 per mouse). At the day cells are ready to be inject, thaw a vial of Basement Membrane Extract (Biotest, 3433-010-01) on ice. Keep 200ul tips in -20° as well.
1w
Once the BME is thawed, trypsinize the cells, count them using trypan blue, and resuspend them in PBS in the concentration of 5M cells in 40ul PBS. add 10ul of BME per 40ul of cells suspension and keep pn ice.
** BME should be always kept on ice
1h
Inject 50ul of cells solution to the lip of each mouse using 300ul syringe.
1h
Allow cells to grow until tumor size reach the size of 4X4 (5-8 days)
1w
Mice treatment
Mice treatment
1d
1d
A day before treatment initiation, suspend GDC-0077 in MCT buffer to reach a dose of 25mg/kg.
Note
average mouse weight 25g. It means 25mg of drug will be sufficient for 40 mice. each mice will be orally gavaged with 200ul of drug solution. So, for example, for 40 mice you will need 25mg of drug and 8ml of MCT buffer
MCT buffer recipe
0.5% Methylcellulose
0.2% tween-80
DI water
Keep The drug on a magnetic stirrer over night.
1d
The next day, gavage each mice with 200ul of the drug solution daily.
Measure tumor size using the caliper at the first day of treatment and every 3-4 days.
Keep treating the mice for 7-10 days, until the effect of the drug can be seen.
Tumor + lymph node collection
Tumor + lymph node collection
Arrange enough plain RPMI medium and ice before start sacrificing mice!
Sacrifice the mice, cage by cage, and collect tumors and mandibular lymph bodes using scissors and forceps. Keep the correct attribution for each set of tumor + lymph nodes. Place everything on ice in the RPMI medium.
Single cell isolation
Single cell isolation
30m
30m
Tumors: Chop 1-2 tumors on a 60mm petri plate using scalpel, until all big chunks are gone.
Collect all the content of the plate using plain RPMI. Once you finished chopping all of the tumors, centrifuge all the tubes, waste the supernatant, and resuspend the tumors in 5ml of an enzymatic dissociation cocktail:
collagenase II 1mg/ml
hyaluronidase 1mg/ml
DNAse I 1mg/ml
Transfer all the content to a gentleMACS™ C Tubes, place the C tubes on the gentleMACS machine, and run an appropriate protocol
Note
In general, for mice solid tumors we use the 37C_m_LIDK_1 protocol
Transfer all the C tube content to a 15 or 50ml tube, centrifuge for 5min on 300g, and waste the supernatant. Resuspend the tumor cells in 4ml RPMI.
Insert a glass pipet to the bottom of the 15ml tube, blocking air tranfer with your thumb. With a syringe, add 2ml of Ficoll-Paque PLUS, making sure not to mix it.
Centrifuge the tubes for 10min, 350g with no brakes.
Collect only the lymphocytes phase to a new 15 ml tube and wash it with PBS. Aspirate the supernatant and resuspend the cells in PBS.
Count the cells and resuspend up to 10^7 cells in 40ul MACS buffer. (same amount for each sample)
Keep the pellet of CD8+ isolation.
MACS buffer
0.5% bovine serum albumin (BSA)
2 mM EDTA
in PBS
Lymphs: Place a 70um strainer on top of a 50ml tube. Place the lymph nodes on top of the strainer together with RPMI. Using the "leg" of a 3ml syringe, press the lymph nodes over the mesh. Collect all the single cells by washing the mesh with RPMI.
Centrifuge for 5min on 300g, and collect the pellet.
Resuspend the cells pellet with 3ml of sterile AKC buffer for 3 min.
Add 9ml of PBS and centrifuge for 5min at 300g. Aspirate the supernatant and resuspend the cells in T-cells medium. Count the cells.
T-cells medium
| Component | Final concentration | Amount | |
| RPMI 1640 | n/a | To 500 mL | |
| L-glutamine | 1x | 5 mL | |
| Penicillin-streptomycin solution (10×) | 1× | 5 mL | |
| 2-mercaptoethanol (1 M) | 50 μM | 25 μL | |
| FBS | 10% (v/v) | 50 mL | |
| HEPES 1M | 25mM | 12.5 mL | |
| sodium pyruvate | 1x | 5 mL | |
| MEM non-essential amino acids | 1x | 5 mL |
ACK Lysis Buffer
| Reagent | Quantity (for 1000 mL) | Final concentration | |
| NH4Cl | 8.02 g | 150 mM | |
| KHCO3 | 1 g | 10 mM | |
| Na2EDTA | 37.2 mg | 0.1 mM |
CD8+ isolation
CD8+ isolation
Following the CD8a+T Cell Isolation Kit mouse Order no. 130-104-075 protocol of Miltenyi :
Add 10 µL of Biotin-Antibody Cocktail per 10⁷ total cells.
Mix well and incubate for 5 minutes in the refrigerator (2−8 °C).
Add 30 µL of buffer per 10⁷ total cells.
Add 20 µL of Anti-Biotin MicroBeads per 10⁷ total cells.
Mix well and incubate for 10 minutes in the refrigerator (2−8 °C).
Proceed to subsequent magnetic cell separation.
▲ Note: A minimum of 500 µL is required for magnetic separation. If necessary, add buffer to the cell suspension.
Place LS Column in the magnetic field of a suitable MACS Separator. For details refer to the respective MACS Column data sheet.
Prepare column by rinsing with 3 mL of buffer.
Apply cell suspension onto the column. Collect flow-through containing unlabeled cells, representing the enriched CD8a+ T cells.
Wash column with 3 mL of buffer. Collect unlabeled cells that pass through, representing the enriched CD8a+ T cells, and combine with the effluent from step 21.
Remove column from the separator and place it on a suitable collection tube. Pipette 5 mL of buffer onto the column. Immediately flush out the magnetically labeled nonCD8a+ T cells by firmly pushing the plunger into the column.
Centrifuge both labeled and unlabeled cells 5min at 300g, and resuspend both in 1ml of T-cell medium. Take 50ul of each for the Flow cytometry validation.
Analyze positive population by Flow
Analyze positive population by Flow
Fc-block cells with 25ul TruStain fcX™ (anti-mouse CD16/32) fc blocker(1:1000) diluted in FACS buffer for 5-10 minutes on ice.
FACS buffer
1% FCS
1mM EDTA
0.05% Na-Azid
in PBS
Add 25ul of appropriately conjugated fluorescent primary antibodies at predetermined optimum concentrations (1:300 by default) and incubate on ice for 15-20 minutes in the dark.
** dilute antibodies in FACS buffer. It is recommended to create a stock solution of each antibody following the next calculation:
2ul of AB stock will be diluted in a total of 25ul, so stock should be 12.5X. 300/12.5 = 24. If we take 1ul of an antibody and dilute it with 23ul of FACS buffer, we then create a stock we can use 2ul of it per sample. This will allow us to mix up to 12 antibodies and no exceeding the 25ul.
prepare DAPI solution by diluting DAPI in FACS buffer to a concentration of 3ug/ml, and add 100ul of this solution per sample.
Analyze the positive and negative population over the Flow cytometer.
CD8+ T cells RNA isolation
CD8+ T cells RNA isolation
Spin cells for 5 min at 300 X g. Remove media and resuspend cells in ice cold PBS. Pellet cells by spinning at 300 X g for 5 min. Lyse cells with TRIZOL Reagent by repetitive pipetting or by passing through syringe and needle. Use 1 ml of the reagent per 5-10 X 10^6 of animal cells.
Incubate the homogenized sample for 5 minutes at room temperature to permit the complete dissociation of nucleoprotein complexes. Centrifuge to remove cell debris. Transfer the supernatant to new tube.
Add 0.2 ml of chloroform per 1 ml of TRIZOL Reagent. Cap sample tubes securely.
Vortex samples vigorously for 15 seconds and incubate them at room temperature for
2 to 3 minutes.
Centrifuge the samples at no more than 12,000 x g for 15 minutes at 2
to 80C.
Note
Following centrifugation, the mixture separates into lower red, phenolchloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains
exclusively in the aqueous phase
Transfer upper aqueous phase carefully without disturbing the interphase into fresh tube. Measure the volume of the aqueous phase (The volume of the aqueous phase is about 60% of the volume of TRIZOL Reagent used for homogenization).
Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5
ml of isopropyl alcohol per 1 ml of TRIZOL Reagent used for the initial homogenization.
Incubate samples at 15 to 30oC for 10 minutes and centrifuge at not more than 12,000
x g for 10 minutes at 2 to 4oC. The RNA precipitate, often invisible before
centrifugation, forms a gel-like pellet on the side and bottom of the tube.
Remove the supernatant completely. Wash the RNA pellet once with 75% ethanol,
adding at least 1 ml of 75% ethanol per 1 ml of TRIZOL Reagent used for the initial
homogenization. Mix the samples by vortexing and centrifuge at no more than 7,500 x
g for 5 minutes at 2 to 8 oC. Repeat above washing procedure once. Remove all
leftover ethanol.
Air-dry or vacuum dry RNA pellet for 5-10 minutes. Do not dry the RNA pellet by
centrifuge under vacuum. It is important not to let the RNA pellet dry completely as
this will greatly decrease its solubility. Partially dissolved RNA samples have an A260/A280
ratio < 1.6. Dissolve RNA in DEPC-treated water by passing solution a few times
through a pipette tip.
CFSE staining of lymph cells
CFSE staining of lymph cells
Prepare a 2 µM working solution by diluting 1 µL of 5 mM CFSE stock solution in 2.5 mL PBS
Spin down and resuspend cells at 10-100 x 10^6 cells/mL in the CFSE working solution.
Incubate cells for 20 minutes at room temperature and keep protected from light.
Quench the staining by adding 5 times the original staining volume of cell culture medium containing 10% FBS.
Pellet cells and resuspend in pre-warmed cell culture medium.
Count the cells and dilute them to 200k/100ul in T-cells medium.
Incubate peptides with lymph node cells
Incubate peptides with lymph node cells
The 24 peptides are in a stock concentration of 2mM. Dilute the peptides 1:100 in T-cells medium and place them according to the map below :
| 1 | 2 | 3 | 4 | 5 | |
| A | |||||
| B | 1 | 7 | 13 | 19 | |
| C | 2 | 8 | 14 | 20 | |
| D | 3 | 9 | 15 | 21 | |
| E | 4 | 10 | 16 | 22 | |
| F | 5 | 11 | 17 | 23 | |
| G | 6 | 12 | 18 | 24 | |
| H |
| 1 | 2 | 3 | 4 | 5 | |
| A | |||||
| B | PMA+ion | 1 | 7 | 13 | 19 |
| C | No peptide | 2 | 8 | 14 | 20 |
| D | No peptide | 3 | 9 | 15 | 21 |
| E | PMA+ion | 4 | 10 | 16 | 22 |
| F | No peptide | 5 | 11 | 17 | 23 |
| G | No peptide | 6 | 12 | 18 | 24 |
| H |
| 1 | 2 | |
| A | ||
| B | ||
| C | ||
| D | ||
| E | ||
| F | ||
| G | ||
| H |
Seed the cells from step 42 in the 24 wells of the peptide + 2 No peptides well + PMA+ION well as a positive control.
Allow cells to grow 48-72 hours. Quantify CFSE together with CD8+ using FACS.