Isolation of senescent cells by iodixanol (OptiPrep) density gradient‐based separation

Abstract

Objectives Chemotherapeutic drugs induce senescence in cancer cells but, unlike replicative senescence or oncogene‐induced senescence, do so rather inefficiently and depending on DNA damage. A thorough understanding of the biology of chemotherapy‐induced senescent cells requires their isolation from a mixed population of adjacent senescent and non‐senescent cancer cells. Materials and methods We have developed and optimized a rapid iodixanol (OptiPrep)‐based gradient centrifugation system to identify, isolate and characterize doxorubicin (DXR)‐induced senescent hepatocellular carcinoma (HCC) cells (HepG2 and Huh‐7) in vitro. Results After cellular exposure to DXR, we used iodixanol gradient‐based centrifugation to isolate and re‐plate cells on collagen‐coated flasks, despite their low or null proliferative capacity. The isolated cell populations were enriched for DXR‐induced senescent HCC cells, as confirmed by proliferation arrest assay, and β‐galactosidase and DNA damage‐dependent γH2A.X staining. Conclusions Analysing pure cultures of chemotherapy‐induced senescent versus non‐responsive cancer cells will increase our knowledge on chemotherapeutic mechanisms of action, and help refine current therapeutic strategies.

HologLev: A Hybrid Magnetic Levitation Platform Integrated with Lensless Holographic Microscopy for Density-Based Cell Analysis

“…In our platform, dead cells show high density profiles (1.15 g/mL) than the live cells. The increase in the density profile of dead cells was used for live/dead separation using density gradient centrifugation . We used density gradient centrifugation to separate high-density dead cells (>1.15 g/mL).…”

Isolation of bacteria from artificial bronchoalveolar lavage fluid using density gradient centrifugation and their accessibility by Raman spectroscopy

“…During centrifugation, the sample is centrifuged through the gradient, and the individual particles remain at the gradient density level that matches their own density. Frequently used solutions are Percoll [ 29 ], Ficoll [ 30 ], sucrose [ 31 ] and OptiPrep [ 32 ], the last of which was used in this study. Therefore, two different solutions were used and mixed together.…”

Nociceptin/orphanin FQ opioid receptor (NOP) selective ligand MCOPPB links anxiolytic and senolytic effects

“…We next tested the senolytic effects of MCOPPB on doxorubicin (DOX)-induced senescent cancer cells, a model of chemotherapy-induced senescence. To this aim, we used two well-established hepatocellular carcinoma (HCC) cell lines (HepG2, Huh-7) [27,43]. DOX was administered at 100 nM for 24 h followed by 6 days of washout, as previously described [27,43], before MCOPPB administration for an additional 24 h. Figure 1C shows the dose-dependent effect of MCOPPB on HepG2 and Huh-7 cell viability, identifying the dose 0.5 µM as the maximal cytostatic dose, without displaying cytotoxic effects.…”

“…To this aim, we used two well-established hepatocellular carcinoma (HCC) cell lines (HepG2, Huh-7) [27,43]. DOX was administered at 100 nM for 24 h followed by 6 days of washout, as previously described [27,43], before MCOPPB administration for an additional 24 h. Figure 1C shows the dose-dependent effect of MCOPPB on HepG2 and Huh-7 cell viability, identifying the dose 0.5 µM as the maximal cytostatic dose, without displaying cytotoxic effects. To assess the rate of cellular senescence at the end of DOX treatment, we used C12FDG (a fluorogenic substrate Fig.…”