Atuune nxt flow cytometer

Flow cytometry was used to quantify the percentage of CSC markers expression in HT-29 and Caco-2 spheroid cells compared to parental cells. The parental and spheroid cells from each cell line were dissociated with trypsin/EDTA and were washed with PBS twice. The dissociated cells were counted using Trypan blue exclusion assay, and if cell viability was more than 95%, they were evaluated for CSC markers expression. The following antibodies were used: anti-CD44 (1:30), anti-CD133 (1:300), anti-CD166 (1:90) (all from abcam, USA). All antibodies were incubated with 3 × 10⁵ cells for 30 min at 4 °C. Goat anti-rabbit IgG-FITC (1:100) (Santa Cruz biotechnology, USA) was used as secondary antibody. The percentage of CSC marker positive cells were evaluated using an Atuune NxT flow cytometer (Thermo Fisher Scientific, USA) and data were analyzed using FlowJo VX software.

DCs were stained with a panel of antibodies for expression of cellular surface molecules and then analysed by flow cytometry. The following fluorochrome‐labelled antibodies were used: PE anti‐human CD40 and CD83, APC anti‐human CD86 and HLA‐DR, Alexa Fluor 488 anti‐human CD14 and PerCP/Cy5.5 anti‐human CD1a (all from Biolegend, USA) for 30 minutes at 4°C. Anti‐mouse Ig, κ/Negative control compensation particles set (BD, USA) was used to optimize fluorescence compensation settings.
To evaluate the expression of putative CSC markers on spheroid cells, anti‐CD44 (abcam, USA), anti‐CD133 (abcam, USA), anti‐CD166 (abcam, USA) and anti‐DCAMKL1 (abcam, USA) primary antibodies and FITC‐goat anti‐rabbit IgG (Santa Cruz biotechnology, USA) secondary antibody were used. The tubes were run on an Atuune NxT flow cytometer (Thermo Fisher Scientific, USA) and analysed using FlowJo VX software.