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.
1. Preparation of target cells that express red fluorescent protein in their nuclei
2. Isolation of suppressor cells from the tumors in mice
NOTE: In this protocol, suppressor cells (i.e., metastasis-associated macrophages (MAMs) and monocytic-myeloid-derived suppressor cells (M-MDSCs)) are isolated from the lung containing metastatic tumors established by E0771-LG cells. Conditions for tissue dissociation and cell sorting should be optimized to isolate the cells from different tissues.
3. Isolation of CD8+ T cells from the spleen of mice
4. Activation and expansion of the isolated CD8+ T cells
5. Setup of co-culture of target cells with pre-activated CD8+ T cells
6. Imaging of the cells
NOTE: Detailed image acquisition settings will vary with the microscope and fluorophores used; the following general acquisition parameters should be employed for optimal 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). (B) Purity of the sorted M-MDSCs (CD45+Ly6G–CD11b+Ly6Chigh) and MAMs (CD45+Ly6G–CD11b+Ly6Clow).
Figure 2: Identification of apoptotic target cells distinct from apoptotic effector cells. Top row: Image acquisition in the red channel allows the identification of target cell nuclei by target detection mask (pink analysis mask). Middle row: Images acquired in the green channel indicate apoptotic effector and target cells. A size-restricted apoptosis mask (teal analysis mask; greater than 80 µm2) allows single apoptotic effector cells to be excluded from the analysis. Bottom row: composite images merged red and green channels with phase contrast image (left) or red/green overlap mask (right). Identification of co-localized, size-restricted green fluorescence with red fluorescence (yellow analysis mask), allows more accurate detection of apoptotic target nuclei (yellow arrowhead) by excluding aggregates of apoptotic effector cells (white arrowheads).
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 |