Apc mouse igg2b

For the analysis of CD24 and CD44 expression on cell membrane, 5 × 10⁵ of cells were collected in Cell Staining Buffer (BioLegend, #420201) and stained with PE-Cy™7-conjugated anti-CD24 (1:100 for 20 min; BD Biosciences, #561646) and APC-conjugated anti-CD44 antibody (1:60 for 20 min; BD Biosciences, #559942) by using PE-Cy™7 Mouse IgG2a (1:100 for 20 min; BD Biosciences, #552868) and APC Mouse IgG2b (1:60 for 20 min; BD Biosciences, #555745) as control staining. Stained cells were analyzed by BD FACSCanto II cytometer and data were acquired by BD FACSDiva v8.0.2 software and processed by FlowJo v10.7.1 software (BD Biosciences). ALDEFLUOR assay was carried out using the ALDEFLUOR assay kit (STEMCELL Inc., #101700) according to the manufacturer’s instructions. The ALDH1 inhibitor, diethylaminobenzaldehyde (DEAB), was used as a negative control. The processed cells were evaluated by BD FACSCanto II cytometer and data were acquired by BD FACSDiva v8.0.2 software and analyzed by FlowJo v10.7.1 software (BD Biosciences).

Mammospheres were grown, treated with C3, collected and dissociated into single cells as before. Then, the cells were washed in cold fluorescence-activated cell sorting buffer (FACS, 2% FBS, 0.05% sodium azide in DPBS), counted and 1 × 10⁶ cell/mL FACS was prepared. The antibodies used to study the expression of CD44 and CD24 markers were as follows: APC mouse anti-human CD44 (BD Pharmingen™, BD Biosciences, CA, USA), APC mouse IgG2b, κ isotype control (BD Pharmingen™, BD Biosciences, CA, USA), PE mouse anti-human CD24 (BD Pharmingen™, BD Biosciences, CA, USA) and PE mouse IGg2a, κ isotype control (BD Pharmingen™, BD Biosciences, CA, USA). APC-Cy7 (100 µL per tube) was used to select for live cells. Incubation with antibodies or isotype controls was for 30 min in darkness at 4 °C. Cells were washed twice in 1 mL FACS buffer and resuspended in 100 µL of FACS buffer. Expression of ALDH was studied using ALDEFLUOR™ Kit (Stem Cell Technologies), based on the manufacturer’s protocol. An aliquot of 1 × 10⁵ cells/tube was prepared on ice for 30 min staining with 20 µL antibody or isotype control. Prior to analysis, the cells were washed twice in 1 mL FACS buffer and resuspended in 100 µL of FACS buffer. The experiments were performed in triplicate and the results are expressed as mean ± SD.