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). (<…

Divulgazioni

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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Citazione di questo articolo
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).

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