Anti cd44 apc

Unpurified splenocytes or isolated subpopulations were subjected to flow cytometric analyses to validate the quality of the cells. Prior to staining, the Fc receptors were blocked by preincubation with anti‐CD16/CD32 antibodies (Biolegend, San Diego, CA, USA) for 10 minutes on ice. Surface staining was accomplished by incubating the cells with fluorochrome‐conjugated specific antibodies for 20 minutes in the dark on ice. The following antibodies (all purchased from Miltenyi Biotec) were employed: anti‐CD3‐FITC (130‐102‐496), anti‐CD4‐FITC (130‐102‐541), anti‐CD4‐PE (130‐102‐619), anti‐CD8‐PE (130‐102‐595), anti‐CD19‐FITC (130‐092‐042), anti‐CD25‐APC (130‐102‐787), anti‐CD44‐APC (130‐102‐563), anti‐CD62L‐PE (130‐102‐907).
Intracellular staining of FoxP3 was performed using an anti‐FoxP3‐PE antibody (130‐098‐119; Miltenyi Biotec) and the FoxP3 Staining Buffer Set (130‐093‐142; Miltenyi Biotec) following the given instructions.
Flow cytometric analyses were run on a FACS Verse (BD Biosciences) or FACS Calibur (BD Biosciences). A total of 10 000 events per sample were acquired and data were evaluated using the FACS Suite or CellQuest Pro software (both BD Biosciences).

Cells were washed twice in PBS and pelleted in a 96- well plate. For the conjugated antibodies cells were stained in 50 μl of flow buffer (0.1% BSA in 1 × PBS) for 30 min at 4 °C in the dark. Cells were washed twice in flow buffer and resuspended in 100 μl of flow buffer prior to reading. We used the following antibodies at the dilutions indicated by the manufacturer: anti-CD49d PE-Violet 770 (Miltenyi Biotec), anti-CD36 Violet Blue (Miltenyi Biotec), anti-CD71 APC (Miltenyi Biotec), anti-basigin FITC (Invitrogen), anti-CD44 APC (Miltenyi Biotec), anti-CD55 APC (Miltenyi Biotec). Anti-AQP1 PE (B-11; Santa Cruz Biotechnology) was used at a 1:100 dilution. To measure ART4 expression, cells were stained with anti-DO3 eluate at a 1:2 dilution for 30 min at 4 °C in the dark and recognized with anti-human IgG Alexa Fluor 647 (life technologies) at a 1:200 dilution for 1 h 4 °C in the dark. Cells were analyzed by using a MACSQuant analyzer 10 flow cytometer (Miltenyi Biotec) and data were analyzed by using the software FlowJo, version 10.4. 50,000 cells were acquired for each sample and cell populations were separated by a live/dead Propidium iodide stain.

Cells were dissociated by treatment with Accutase (Invitrogen), resuspended in 1% bovine serum albumin (BSA) (Sigma‐Aldrich) in phosphate‐buffered saline (PBS) and incubated for 15 minutes at 4°C with anti‐CD271 (p75NTR)‐PE, anti‐SOX1‐PE, anti‐CD44‐APC, anti‐CD73‐PE or anti‐CD105‐APC (all from Miltenyi Biotec, Bergisch Gladbach, Germany). The cells were washed once in 1% BSA in PBS and analysed using a BD FACSCalibur (BD Biosciences). Isotype control antibodies (Miltenyi Biotec) were used as negative controls. p75high and p75low cells were separately sorted using BD FACSAria™ III Cell Sorter (BD Biosciences).