Phycoerythrin pe

MC and SC neonatal astrocytes from WT or SOD1^G93A pups were detached by trypsin-EDTA and centrifuged for 5 min at 500× g. About 5 × 10⁵ cells were resuspended and saturated for unspecific bonds by incubating with 0.5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS, pH 7.4) for 15 min at RT. Aliquots of the suspension were stained (1 h at RT) with the following fluorochrome-conjugated antibodies for flow cytometry: mouse monoclonal anti-GFAP antibody conjugated with Alexa Fluor A488 (Thermo Fisher Scientific, Cat# 53-9892-82), rat monoclonal anti-ACSA2 antibody conjugated with phycoerythrin (PE) (Miltenyi Biotec, Cat# 130-102-365) and rat monoclonal anti-TMEM119 antibody conjugated with Alexa Fluor A488 (Abcam, Cat# ab225497). For GFAP staining, cell suspensions were previously fixed and permeabilized, 20 min at 4 °C, by using the Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Bioscience, Cat# 554714). After staining, cells were centrifuged (5 min at 500× g) and pellets were resuspended in PBS for flow cytometry analyses. Cell debris and dead cells were excluded from analysis by 7-aminoactinomycin D (7-AAD) labeling. Data were acquired on a Guava easyCyte 6 flow cytometer (Merck Millipore, Burlington, MA, USA) and processed using the GuavaSoft 3.1.1 software (Merck Millipore).

Cell cycle distribution by PI staining and apoptosis by AnnexinV-FITC (BD biosciences, San Diego, CA, USA)/PI staining were performed as previously described [62 ]. For analysis of CD133+ subpopulation, the cells were washed with PBS, and then incubated with antibody against CD133/1 conjugated with phycoerythrin (PE; MiltenyiBiotec, BergischGladbach, Germany) on ice in the dark for 20 min. After the cells had been washed with PBS, the CD133⁺ and CD133⁻ populations were analyzed by FACS. Active caspase-3 Apoptosis Kit (BD biosciences) was used to detect the heterodimer of 17 and 12 kDa subunits, which is derived from the pro-enzyme. For annexinV and caspase-3 double staining, cells were washed with 1× binding buffer and then incubated with FITC annexinV (BD biosciences) for 15 min at room temperature in the dark. After annexinV staining, caspase-3-PE staining was performed on the same cells following manufacturer instructions. ALDH activity was evaluated by ALDEFLUOR kit (ALDH, STEMCELL Tecnologies, Vancouver, BC, Canada) following manufacturer instructions. All flow cytometric analyses were performed by using BD Accuri™ C6 flow cytometer (BD biosciences).