Flow Cytometry-Based Quantification of Therapy-Induced Senescent Cancer Cells
Published: March 29, 2024
Abstract
Source: Flor, A., et al. Far-Red Fluorescent Senescence-Associated β-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry. J. Vis. Exp. (2022)
This video illustrates a flow cytometry-based assay for quantifying therapy-induced senescent cancer cells with dual senescence markers. The detection and quantification of senescent cells involve using a β-galactosidase substrate. The substrate is cleaved by active lysosomal β-galactosidase within the senescent cells, producing a fluorescent product that, in conjunction with autofluorescent lipofuscin granules, marks the senescent cancer cells.
Protocol
1. Induction of senescence by chemotherapy drugs in cultured cancer cells NOTE: All cell manipulation steps in this section should be performed in a biosafety cabinet using sterile practices. This section is written for adherent cell types. Suspension cells may be used with appropriate modifications as noted. Grow cancer cell line(s) according to the standard protocol from the supplier or the laboratory that provided the specific cell line(s) used….
Declarações
The authors have nothing to disclose.
Materials
| Bafilomycin A1 | Research Products International | B40500 | |
| Bleomycin sulfate | Cayman | 13877 | |
| Bovine serum albumin (BSA) | US Biological | A1380 | |
| Calcein Violet 450 AM viability dye | ThermoFisher Scientific | 65-0854-39 | eBioscience |
| DPP4 antibody, PE conjugate | Biolegend | 137803 | Clone H194-112 |
| Cell line: A549 human lung adenocarcinoma | American Type Culture Collection | CCL-185 | |
| Cell line: B16-F10 mouse melanoma | American Type Culture Collection | CRL-6475 | |
| Cell scraper | Corning | 3008 | |
| Cell strainers, 100 µm | Falcon | 352360 | |
| DDAO-Galactoside | Life Technologies | D6488 | |
| DMEM medium 1x | Life Technologies | 11960-069 | |
| DMSO | Sigma | D2438 | |
| DNAse I | Sigma | DN25 | |
| Doxorubicin, hydrochloride injection (USP) | Pfizer | NDC 0069-3032-20 | |
| Doxorubicin, PEGylated liposomal (USP) | Sun Pharmaceutical | NDC 47335-049-40 | |
| EDTA 0.5 M | Life Technologies | 15575-038 | |
| Etoposide | Cayman | 12092 | |
| FBS | Omega | FB-11 | |
| Fc receptor blocking reagent | Biolegend | 101320 | Anti-mouse CD16/32 |
| Flow cytometer (cell analyzer) | Becton Dickinson (BD) | Various | LSRFortessa |
| Flow cytometer (cell sorter) | Becton Dickinson (BD) | Various | FACSAria |
| GlutaMax 100x | Life Technologies | 35050061 | |
| HEPES 1 M | Lonza | BW17737 | |
| Liberase TL | Sigma | 5401020001 | Roche |
| Paraformaldehyde 16% | Electron Microscopy Sciences | 15710 | |
| Penicillin/Streptomycin 100x | Life Technologies | 15140122 | |
| Phosphate buffered saline (PBS) 1x | Corning | MT21031CV | Dulbecco's PBS (without calcium and magnesium) |
| Rainbow calibration particles, ultra kit | SpheroTech | UCRP-38-2K | 3.5-3.9 µm, 2E6/mL |
| RPMI-1640 medium 1x | Life Technologies | 11875-119 | |
| Sodium chloride 0.9% (USP) | Baxter Healthcare Corporation | 2B1324 | |
| Software for cytometer data acquisition, "FACSDiva" | Becton Dickinson (BD) | n/a | Contact BD for license |
| Software for cytometer data analysis, "FlowJo" | TreeStar | n/a | Contact TreeStar for license |
| Trypsin-EDTA 0.25% | Life Technologies | 25200-114 |
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Citar este artigo
Flow Cytometry-Based Quantification of Therapy-Induced Senescent Cancer Cells. J. Vis. Exp. (Pending Publication), e22062, doi: (2024).