Apc conjugated cd44 antibody

For cell-surface analysis, cells were harvested by dissociation using 0.05% trypsin/EDTA. A total of 1 × 10⁶ cells were resuspended in 200 μL PBS with 1% FBS, incubated with antibodies at the recommended concentrations at 4 °C for 30 min, and then detected by flow cytometry (BD FACSCanto II, BD Biosciences, USA). Data were analyzed using FlowJo software. The antibodies used in flow cytometry assay were as follows: APC-conjugated CD163 antibody (333610, Biolegend), PE-conjugated CD206 antibody (321106, Biolegend), APC-conjugated CD44 antibody (559942, BD Biosciences), PE-conjugated CD24 antibody (555428, BD Biosciences).

Cells were detached and incubated with APC-conjugated CD44 antibody (BD-PharMingen, San Diego, CA, USA 559942) or isotype control APC-conjugated IgG2B (BD-PharMingen) in PBS BSA 1% (Sigma) for 30 minutes on ice prior to flow cytometric analysis. Sytox Blue Stain (Life Technologies, Eugene, OR, USA S34857) was added to exclude dead cells. For CD44v8-10 staining 1×10⁵ cells were incubated with 3 μg/ml anti-human CD44v9 primary antibody (clone: RV3) (Cosmo Bio Co. Ltd, Tokyo, Japan LKG-M001) in PBS 0.2 % BSA for 45 minutes at 4°C. Cells were then incubated with the secondary antibody APC-labeled Goat anti-Rat IgG (H+L) (Invitrogen, Carlsbad, CA, USA A10540) for 30 minutes at 4°C. After washing, cells were assayed using a CyAn ADP flow cytometer (Beckman Coulter, Brea, CA, USA) and data analyzed employing FCS5 express Software (De Novo Software). For cell cycle analysis, after labeling for CD44v9, the cells were fixed with 70% ethanol, a few drops at a time mixing the cells, and incubated on ice for 30 minutes. The cells were centrifuged at 500 x g for 10 minutes, washed once in PBS by centrifugation and resuspended in 1 ml PBS containing 5 μg/ml of propidium iodide. Samples were assayed by CyAn ADP flow cytometer and analyzed employing FCS5 express Software.

Cells to be analysed were trypsinised, washed and incubated for 1 h in antibody diluted 1:100 in PBS. Flow cytometry was performed on an Accuri Flow Cytometer (BD Biosciences) and analysis of results was performed using a FlowJo software package. APC-conjugated CD44 antibody was purchased from BD Pharmingen, PE-conjugated ALDH1 antibody was purchased from Stratech.