Brdu cell proliferation assay

Cell viability was analyzed by Cell Titer-Glo (CTG; Promega), and S-phase DNA synthesis by BrdU Cell Proliferation assay (Cell Signaling Technology). For colony-forming assay, 200 cells were plated in triplicate in 1 ml of mixture composed of 1.1% methylcellulose (MethoCult^TM STEMCELL Technologies) in RPMI-1640 + 10% FBS. Crystal violet-stained colonies were scored after 2 weeks under an inverted microscope (Leica DM IL LED) at ×5 magnification. Cell migration was analyzed by Transwell migration assay (BD Biosciences) as described [16 (link)].

Cell proliferation was measured using the BrdU cell Proliferation Assay (Cell Signaling Technology), according to the manufacturer’s instructions. Proliferation values were measured using a microplate photometer (Thermo Fisher Scientific). Absorbance values were measured at 450 nm.

Cell proliferation was assessed as described (Tsitsipatis et al., 2021 (link)). Briefly, 2 × 10⁴ cells were plated per well and proliferation rates were assessed using both cell counting and BrdU incorporation up to day 6. For cell counting, cells were harvested and washed once with 1× PBS (Gibco), and total cells were counted on an automatic cell counter (Bio‐Rad). For BrdU incorporation, BrdU Cell Proliferation Assay (Cell Signaling Technology) was employed following the manufacturer's instructions. Briefly, BrdU was added one day after seeding and the incorporation was detected for up to day 6 by reading at 450 nm on a GloMax Explorer plate reader (Promega).

The viability of ex vivo expanded lymphocytes was first verified by trypan blue dye exclusion (Sigma Aldrich, St. Louis, MO, USA), followed by propidium iodide and annexin V staining (Sigma Aldrich). The quantitative cell proliferation was performed and reported based on BrdU cell proliferation assay (Cell Signaling Technology, Danvers, MA, USA) as per manufacturer’s protocol.

HT29 cells were seeded in 24-well plates at a density of 1 × 10⁵ cells/well (for qRT-PCR analysis) and in 96-well plates at 20,000 cells per well (for viability/proliferation assays) in McCoy’s 5A Medium Modified culture medium and co-cultured with polysorbate 80 (p80) at a concentration of 0.25% w/v diluted in the same media. HT29 cells were cultured in 0.25% p80 for 1, 2, 4, and 6 hours (h) and cells collected at each time point, washed with phosphate-buffered saline (PBS, Sigma), and lysed, with lysates frozen at −80°C for PCR analyses. All conditions were performed in triplicates. For RNA extraction, the triplicates of each condition were extracted and pooled for gene expression analysis. At the 6 h time point, HT29 cells treated with 0.25% p80 were assayed for cell proliferation (BrdU Cell Proliferation Assay, Cell Signaling Technology, Ireland) and cell viability (CellTiter-Blue, Viability Assay, Promega Corporations, MyBio, Ireland) following the manufacturer’s instructions.

The in vitro viability assay WST-1 [Cleavage of the tetrazolium salt WST-1 (4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) to formazan] was performed according to manufacturer’s instructions (Clontech, Mountain view, USA). Cells were plated at a density of 10⁶ cells/ml and incubated for 24 hours (h) at 37 °C. Plates were read at 450 nm wavelength on a DAS plate reader (Rome, Italy). Results from WST-1 viability assays are expressed as fraction of treated versus untreated cells. Trypan blue exclusion, as well as Annexin V and propidium iodide (PI) methods were used to determine cell death. The BrdU Cell Proliferation Assay which detects 5-bromo-2′-deoxyuridine (BrdU) incorporated into cellular DNA during cell proliferation using an anti-BrdU antibody, was performed according to manufacturer’s instructions (Cell signaling Technology, Danvers, U.S). Cells were plated at a density of 10⁶ cells/ml and incubated for 24 hours (h) at 37 °C. Plates were read at 450 nm wavelength on a DAS plate reader (Rome, Italy).

For determining cell viability, 50,000 MDA-MB-231 cell variants were plated into a 6-well plate. At 72 h, cells were trypsinized, harvested, stained with trypan blue, and counted using a hemocytometer. Cells were deemed viable upon trypan blue exclusion. The Cell Proliferation Kit I MTT Assay (Millipore Sigma, St. Louis, MO, USA) was also used to evaluate tumor cell viability over time. 2000 MDA-MB-231, MDA-MB-468, or MVT1 variants were plated and measurements taken at 72 h using a Promega Glomax Discover microplate reader. For DMSO, BQU57, and Taxol treatments, the media contained drugs at the indicated concentration and measurements were taken after 72 h.
The BrdU Cell Proliferation Assay (Cell Signaling, Danvers, MA, USA) was used to evaluate tumor cell proliferation. 10,000 MDA-MB-231 or 5000 MVT1 cell variants were plated and the assay was performed following manufacturer’s instructions. At 72 h, absorbance was read at 450 nm using a Promega Glomax Discover microplate reader.
The Dead Cell Apoptosis Kit with Annexin V Alexa Fluor™ 488 (Invitrogen, Carlsbad, CA, USA) was used to evaluate apoptotic populations. MDA-MB-231 or MVT1 cell variants were plated and allowed to reach ~90% confluency. Floating and harvested cells were collected and treated according to manufacturer’s instructions. Cells were acquired using a LSR II.