Cd44 phycoerythrin pe

The analysis of CD20 and CD44 surface markers and the ALDH1 activity were performed using a Becton Dickinson FACSCanto II Flow Cytometer from the CIC Scientific Instrumental Centre (University of Granada) as previously described [29]. Briefly, ALDEFLUOR assays (Stem Cell Technologies, Vancouver, BC, Canada) to detect ALDH1 activity in viable cells were performed according to the manufacturer's instructions. Cells were suspended in ALDEFLUOR assay buffer containing ALDH1 substrate (BAAA, 1 μmol·L⁻¹ per 1 × 10⁶ cells) and incubated for 45 min at 37 °C in darkness. Diethylaminobenzaldehyde (DEAB) was used as an ALDH1 inhibitor to set ALDH1 gates. The bright fluorescent ALDH1‐expressing cells were detected in the green fluorescent channel (520–540 nm). Cell surface levels of CD44 and CD20 were determined with anti‐human antibodies CD44‐phycoerythrin (PE) and CD20‐allophycocyanin (APC) (Miltenyi Biotec, Bergisch Gladbach, Germany), respectively. The bright fluorescent PE and APC were detected in red (564–606 nm) and blue (650–670 nm), respectively. All samples were analysed on a FACSCanto II (BD Biosciences, San Jose, CA, USA) using the facsdiva software.

Cells were trypsinized, washed in PBS, and stained with the following anti-mouse antibodies: CD44-phycoerythrin (PE; cat:130-102-606), CD45-PE (cat:130-102-781), F4/80-fluorescein isothiocyanate (cat:130-102-327), and Sca-1-allophycocyanin (cat:130-102-343) from Miltenyi Biotec. The samples were processed using a NovoCyte flow cytometer (ACEA Biosciences) and analyzed using the NovoExpress software.