Lecture 16: Fluorescence-Activated Cell Sorting (FACS) and Clonogenic Assay PDF

Lecture 16 Dr. Kavitha Thirumurugan Professor HAG, SBST  Fluorescence-activated cell sorting (FACS) Annexin V Apoptosis assay employing FACS analysis was c...

Lecture 16 Dr. Kavitha Thirumurugan Professor HAG, SBST  Fluorescence-activated cell sorting (FACS) Annexin V Apoptosis assay employing FACS analysis was completed MCF-7 breast cancer cells using 3-day treatments at the indicated concentrations of SFN, WA or both compounds (** p < 0.01, *** p < 0.001). The results represent three separately cultured experimental replicates. Numbers on the X-axis indicate compound concentrations. https://www.mdpi.com/1422-0067/18/5/1092  Annexin V Apoptosis assay employing FACS analysis was completed on MDA-MB-231 breast cancer cells using 3-day treatments at the indicated concentrations of SFN, WA or both compounds (** p < 0.01, *** p < 0.001). The results represent three separately cultured experimental replicates. Numbers on the X-axis indicate compound concentrations. https://www.mdpi.com/1422-0067/18/5/1092  Flow Cytometry analysis (A) Negative control group: There was no distinct apoptotic peak. (B) Experimental group: The hypodiploid peak before the G1 peak shown in the cell cycle histogram was the apoptotic peak (Ap peak) formed because of apoptosis cells. https://doi.org/10.12659%2FMSMBR.893327  Flow cytometry is a classical method to detect cell apoptosis with high sensitivity and can be used for simultaneous cell cycle analysis. However, a general laboratory may not be equipped with a flow cytometer, and they are costly to run. No significant difference was observed between the ability of flow cytometry and AO/EB staining to detect apoptosis. Therefore, fluorescent staining can feasibly be applied to evaluate apoptosis in osteosarcomas or other malignant tumors. AO/EB is a more economical and convenient method compared with flow cytometry.  Clonogenic assay (or) Colony Forming Assay (CFA) (or) In vitro tumorogenic assay https://www.youtube.com/watch?v=zPeY70vsHlk The colony-forming assay, also known as the clonogenic assay, is an in vitro cell survival assay that evaluates a cell's ability to grow into a colony. The colony is defined to consist of at least 50 cells. The assay tests every cell in the population for its ability to undergo “unlimited” division. Colony forming or clonogenic assay is an in vitro quantitative technique to examine the capability of a single cell to grow into a large colony through clonal expansion.  Clonogenic assay is the method of choice to determine cell reproductive death after treatment with ionizing radiation, but can also be used to determine the effectiveness of other cytotoxic agents. Only a fraction of seeded cells retain the capacity to produce colonies. Before or after treatment, cells are seeded out in appropriate dilutions to form colonies in 1–3 weeks. Colonies are fixed with glutaraldehyde (6.0% v/v), stained with crystal violet (0.5% w/v) and counted using a stereomicroscope.  In house Protocol- Colony Forming Assay of HCT116 (CRC) Day 1- Cell seeding: The cells should be seeded as 10,000 cells/well along with the media Keep control. Wait till the cells get 30%- 40% confluency and the cells retain their morphology. Day 2- Treatment of the cells with various concentrations of drugs (300µl media + drug). Keep the plate at 37°C, 5% CO2 for 3 days. Observe each day. Day 3- Take the plate out, decant the media, and wash with PBS (300µl). Swirl it around gently and decant the PBS. Add Coomassie Blue stain (300μl ) to each well and incubate for 10 minutes. Decant the stain. Observe the plate under the White Illuminator  Clonogenic assay of HCT116 cells https://doi.org/10.1007/s10495-023-01826-4 HCT116 cells (CRC- Colorectal cancer cells) are treated with phloretin at different concentrations. Phloretin is the major dihydrochalcone polyphenol and mainly found in apple, pear, and strawberry. Phloretin is known to reduce the development of CRC by inhibiting type 2 glucose transporters and activating p53-mediated signaling. More than 80% of CRCs are initiated due to adenomatous polyposis coli (APC) mutations correlated with the Wnt activation.  Clonogenic assay of HCT116 cells https://doi.org/10.1007/s10495-023-01826-4 Clonogenic assay of HCT-116 and treated with phloretin (25, 50, 100 200 µM), and stained with 0.5% crystal violet.  Clonogenic assay of HCT116 cells https://doi.org/10.1007/s10495-023-01826-4 Clonogenic assay of HCT-116 and treated with phloretin (25, 50, 100 200 µM), and stained with 0.5% crystal violet. Bar graphs showing relative colony growth of HCT-116 cells. Counting of colonies was done using ImageJ (version1.53e). Data showed as mean± SD, n= 3, *p≤ 0.05, **p≤ 0.01, ***p≤ 0.001, ****p≤ 0.0001 in comparsion to control cells  The clonogenic assay revealed that treatment with phloretin at different concentrations (25, 50 100, and 200 µM) significantly reduced the number of colonies formed by the treated cells in comparison to untreated control cells. 1350 colonies were counted in the control plate and treatment with the highest concentration (200 µM) of phloretin reduced the colony number to 700, (p≤ 0.0001) in HCT-116 cells. The count of colonies clearly showed the ability of phloretin to inhibit the proliferation of HCT-116 cells in a concentration-dependent manner. This finding indicated that phloretin has an inhibitory effect on CRC proliferation.  Flow cytometry results https://doi.org/10.1007/s10495-023-01826-4 To determine the mechanism induced by phloretin in decreasing proliferation of colon cancer cells, progression of cell cycle was studied by flow cytometry. Analysis revealed that after treating cells for 48 h, we observed disrupted pattern of cell cycle which showed accumulation of cells in the G2/M phase (8.21% to 35.33%, p≤0.0001) in HCT-116 cells  Apoptosis https://doi.org/10.1007/s10495-023-01826-4 Bar graphs representing apoptotic cell percentage as observed by Annexin V-FITC/PI assay. Results are presented as the mean percentage of early apoptotic and late apoptotic cells induced by the treatment of phloretin. Data showed as mean± SD, n = 3, *p ≤ 0.05, ****p≤ 0.0001, ##p≤ 0.01, ####p≤ 0.0001 in comparison to control cells  To investigate the potential of phloretin in inducing apoptosis in HCT-116 and SW-480 cells flow cytometry analysis was performed. The results signified that at lower concentrations there was no significant induction of apoptosis but at higher concentrations (100 µM and 200 µM), a significant increase in both early and late apoptotic cells was observed indicating concentration dependent increase in cell death.