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.
The authors have nothing to disclose.
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 |