MBTP 5: Immunophenotyping of tumor-infiltrating CD8+ T cells from murine subcutaneous melanoma using flow cytometry
Miltenyi Biotec-tested panel 5 (MBTP 5)
Miltenyi Biotec-tested panel 5 (MBTP 5)
A sample of a subcutaneous melanoma tumor was dissociated, CD8+ T cells were enriched via MicroBead-based enrichment, and cells were characterized via flow cytometry.
Protocol
This protocol includes detailed instructions for preparing necessary reagents and materials and then carrying out each step.
Labeling of cells with Viobility™ Fixable Dye
- Viobility Fixable Dye was reconstituted in advance. The vial was warmed up to room temperature to avoid water condensation. One vial of lyophilized Viobility Fixable Dye was reconstituted by addition of 100 µL anhydrous DMSO to the dye vial and mixed until completely dissolved. The solution was aliquoted and stored at –20 °C under anhydrous (desiccant) conditions and protected from light for up to one month.
- Volumes given below were for up to 107 nucleated cells. When working with fewer than 107 cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g. for 2×107 nucleated cells, use twice the volume of all indicated reagent volumes and total volumes).
- Cell number was determined.
- Cells were washed by addition of 1 mL of 1× PBS and centrifuged at 300×g for 10 minutes. Supernatant was aspirated completely.
- Cells were resuspended in 100 µL of 1× PBS.
- 1 µL of Viobility Fixable Dye was added.
- Samples were mixed well and incubated for 15 minutes in the dark at room temperature.
▲Note: Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice requires increased incubation times. - Cells were washed by addition 1 mL PEB buffer and centrifuged at 300×g for 10 minutes. Supernatant was aspirated completely.
- Step 6 was repeated.
- Surface staining was performed.
Surface staining
- Volumes given below were for up to 106 nucleated cells. When working with fewer than 106 cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g. for 2×106 nucleated cells, use twice the volume of all indicated reagent volumes and total volumes).
- Cell number was determined.
- Cells were centrifuged at 300×g for 10 minutes. Supernatant was aspirated completely.
- A master mix with all antibodies was prepared according to the dilutions mentioned in the respective antibody data sheets.
- 100 µl antibody master mix was added to the cells.
- Cells were resuspended and incubated for 10 minutes in the dark at 4 °C.
- Cells were washed by addition of 1 mL of PEB buffer and centrifuged at 300×g for 5 minutes at 4 °C. Supernatant was aspirated completely.
- Cells were resuspended in 100 µL of PEB buffer and analyzed by flow cytometry using the MACSQuant® Analyzer.
Materials
The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.
Antibody panel details
Antibody panel 1
| Specificity | Clone | Purpose | Fluorochrome | Detection filter (nm) (laser) |
| CD8b | REA793 | Cytotoxic T cells | VioBlue® | 450/50 (violet) |
| CD223 | REA776 | T cell exhaustion | Vio® Bright B515 | 525/50 (blue) |
| TIM-3 | REA602 | T cell exhaustion | PE | 585/40 (blue) |
| CD4 | REA604 | T helper cells | PE-Vio 615 | 655-730 (blue) |
| CD279 (PD1) | REA802 | T cell exhaustion | APC | 655-730 (red) |
| CD44 | REA664 | T cell activation | APC-Vio 770 | 750LP (red) |
| Viobility™ 405/520 Fixable Dye | - | Viability | - | 525/50 (violet) |
Antibody panel 2
| Specificity | Antibody clone | Purpose | Fluorochrome | Detection filter (nm) (laser) |
| CD8b | REA793 | Cytotoxic T cells | VioBlue | 450/50 (violet) |
| CD103 | REA789 | Regulatory T cells | Vio Bright B515 | 525/50 (blue) |
| CD183 (CXCR3) | REA724 | T cell activation | PE | 585/40 (blue) |
| CD4 | REA604 | T helper cells | PE-Vio 615 | 655-730 (blue) |
| CD39 | REA870 | Regulatory T cells | APC | 655-730 (red) |
| CD44 | REA664 | T cell activation | APC-Vio 770 | 750LP (red) |
| Viobility™ 405/520 Fixable Dye | - | Viability | - | 525/50 (violet) |
Additional reagents used
- Phosphate-buffered saline (PBS), pH 7.2
- PEB buffer: PBS, pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA
- Deionized or distilled water
- Flow cytometer, for example, MACSQuant® Analyzer 10 (# 130-096-343) or MACSQuant X (# 130-105-100)
- (Optional) MACSQuant Calibration Beads (# 130-093-607)
- (Optional) MACS® Comp Bead Kit, anti-REA (# 130-104-693)
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130-118-563
TIM-3 Antibody, anti-mouse, PE, REAfinity™
REA602
FC, MICS, IF, IHC
150 µg in 1 mL (1)
EUR 216,00
