An In Vitro Assay for Studying the Cytotoxicity of Pre-Activated CD8+ T Cells against Cancer Cells
Published: January 31, 2024
Abstract
Source: Kitamura, T., et al. Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells. J. Vis. Exp. (2019)
This video showcases an apoptosis assay in cancer cells induced by pre-activated effector T cells. By co-culturing these cells with cancer cells marked with fluorescent nuclei, a caspase-cleavable fluorogenic substrate is added. Apoptotic cancer cells will display a different color in their nuclei, leading to dual fluorescence. This dual fluorescence is indicative of cancer cells undergoing apoptosis.
Protocol
1. Preparation of target cells that express red fluorescent protein in their nuclei Obtain a target mouse cancer cell line from an appropriate source. NOTE: In this protocol, a highly metastatic derivative of E0771 mouse mammary tumor cells (E0771-LG) is used. Parental E0771 cells originate from C57BL/6 mice. Thaw and maintain a vial of E0771-LG cells with Dulbecco's Modified Eagles Medium (DMEM) including 10% (v/v) fetal bovine serum (FBS) in a cell cult…
Representative Results
Figure 1. Gating strategy to isolate suppressor cells from the metastatic lung. (A) Representative dot plots to isolate monocytic myeloid-derived suppressor cells (M-MDSCs) and metastasis-associated macrophages (MAMs). The threshold of Ly6C level to distinguish MAMs (Ly6Clow) and M-MDSCs (Ly6Chigh) is based on that of resident alveolar macrophages (RMAC). (<…
Disclosures
The authors have nothing to disclose.
Materials
| 0.05% Trypsin EDTA (1X) | Gibco | 25300-054 | |
| 12-Well Cell Culture Plate | Freiner Bio-One | 665-180 | |
| 1x PBS | Gibco | 14190-094 | |
| 2-mercaptethanol | Sigma | M6250-10ML | |
| 5mL Plystyrene Round-Bottom Tube | FALCON | 352054 | |
| 96-Well Cell Culture Plate (Round Bottom ) | Costar | 3799 | Co-culture of CD8+T cells with sorted myeloid cells |
| 96-well plate (Flat bottom) | Nunc | 165305 | Co-culture of CD8+T cells with target cells for cytotoxicity assay |
| AF647 anti-mouse F4/80 Antibody | BIO-RAD | MCA497A647 | Clone: CIA3-1, Lot#: 1707, 2 μL/1×10^6 cells |
| AlexaFluor700 anti-mouse CD8 Antibody | Biolegend | 100730 | Clone: 53-6.7, Lot#: B205738, 0.5 μL/1×10^6 cells |
| anti-mouse CD28 Antibody | Biolegend | 102111 | Activation of isolated CD8+ T cells, Clone: 37.51, Lot#: B256340 |
| anti-mouse CD3e Antibody | Biolegend | 100314 | Activation of isolated CD8+ T cells, Clone: 145-2C11, Lot#: B233720 |
| APC anti-mouse CD3 Antibody | Biolegend | 100236 | Clone: 17A2, Lot#: B198730, 0.5 μL/1×10^6 cells |
| APC/Cy7 anti-mouse Ly6C Antibody | Biolegend | 128026 | Clone: HK1.4, Lot#: B248351, 1 μL/1×10^6 cells |
| Bovine Serum Albmin | Sigma | A1470-100G | |
| Cell Strainer (100μm Nylon) | FALCON | 352360 | To smash the spleen |
| Cell Strainer (40μm Nylon) | FALCON | 352340 | To filter the lung digestion |
| DAPI | Biolegend | 422801 | |
| Dulbecco′s Modified Eagle′s Medium | Gibco | 41966-029 | |
| EasySep Mouse CD8+ T Cell Isolation Kit | StemCell Technologies | 19853 | |
| Fetal Bovine Serum | Gibco | 10270-106 | |
| FITC anti-mouse CD4 Antibody | Biolegend | 100406 | Clone: GK1.5, Lot#: B179194, 0.5 μL/1×10^6 cells |
| Geltrex Ready-to-Use | Gibco | A1596-01 | Coating the 96-well plates for cytotoxicity assay |
| IncuCyte NucLight Red Lentivirus Reagent | Essen BioScience | 4476 | Lenti viral particules encoding mKate2 |
| IncuCyte ZOOM | Essen BioScience | Detector (fluorescence microscope) | |
| IncuCyte ZOOM 2018A | Essen BioScience | Analysis software | |
| L-Glutamine (100X) | Gibco | A2916801 | |
| Lung Dissociation Kit | Miltenyi | 130-095-927 | Preparation of single cell suspension from the tumor-bearing lung |
| MycoAlert Mycoplasma Detection Kit | Lonza | LT07-318 | |
| Non-essential amino acid (100X) | Gibco | 11140-035 | |
| NucView488 | Biotium | 10403 | Fluoregenic caspase-3 substrate |
| PE anti-mouse Ly6G Antibody | Biolegend | 127607 | Clone: 1A8, Lot#: B258704, 0.5 μL/1×10^6 cells |
| PE/Cy7 anti-mouse CD11b Antibody | Biolegend | 101216 | Clone: M1/70, Lot#: B249268, 0.5 μL/1×10^6 cells |
| Pen Strep | Gibco | 15140-122 | Penicillin Streptomycin for primary culture of cells |
| PerCP/Cy5.5 anti-mouse CD45 Antibody | Biolegend | 103132 | Clone: 30-F11, Lot#: B249564, 0.5 μL/1×10^6 cells |
| Polybrene (Hexadimethrine bromide) | Sigma | H9268 | |
| Puromycin | Gibco | A11138-03 | |
| RBC Lysis Buffer (10X) | Biolegend | 420301 | |
| Recombinant murine IL-2 | Peprotech | 212-12 | Activation of isolated CD8+ T cells |
| Sodium pyruvate (100X) | Gibco | 11360-070 | |
| TruStain fcX (anti-mouse CD16/32) Antibody | Biolegend | 101320 |
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Cite This Article
An In Vitro Assay for Studying the Cytotoxicity of Pre-Activated CD8+ T Cells against Cancer Cells. J. Vis. Exp. (Pending Publication), e21904, doi: (2024).