Anti cd34 pe

The same cell staining reagents and the same reaction time were used for the CTC-FIND method and the RareCyte method. Specifically, cell staining was performed using a proprietary custom reagent kit developed by RareCyte. This included the following: 4′,6-diamidino-2-phenylindole (DAPI) to stain the cell nucleus) and antibodies specific for the epithelial marker CK, the epithelial marker EpCAM, and the cell surface antigen CD45/CD66b (used for staining leukocytes). We modified the assay by adding additional counter-stain reagent with the following phycoerythrin (PE)-conjugated antibodies: anti-CD14-PE (Clone M5E2, 1:200, BioLegend, San Diego, CA, USA), anti-CD34-PE (Clone 581, 1:200, BioLegend, San Diego, CA, USA), and anti CD11b-PE (Clone M1/70, 1:200, BioLegend, San Diego, CA, USA). For the CTC-FIND method, all staining was done with the slit-filter isolation unit of CTC-FIND. For the RareCyte method, all staining was done with the AutoStainer Link 48 (Agilent Technologies, Santa Clara, CA, USA) and according to manufacturer’s protocol.

The following antibodies were purchased from Biolegend Inc. (San Diego, CA, USA): anti-CD3-pacific blue (17A2), anti-CD4-pacific blue (GK1.5), anti-NK1.1-pacific blue (PK136), anti-CD11b-pacific blue (M1/70), anti-CD11C-pacific blue (N418), anti-Ter-119-pacific blue (Ter-119), anti-CD8-pacific blue (53-6.7), anti-B220-pacific blue (RA3-61B2), anti-CD4-PE (GK1.5), anti-CD4-FITC (GK1.5), anti-CD8-APC (53-6.7), anti-CD8-Pecy7 (53-6.7), anti-CD23-PECy7 (B3B4), anti-CD21/CD35 (CR2/CR)-PerCP/Cy5.5 (7E9), anti-Gr1-Pecy7 (RB6-8C5), Anti-IAb-FITC (KH74), anti-B220-APC-Cy7 (RA3-61B2), anti-CD117-PE-Cy7 (2B8), anti-PDCA1-APC (927), anti-CD135 (flt3)-APC (A2F10), anti-CD25-PE (3C7), anti-CD11C (N418), anti-IgD-PerCP/Cy5.5 (11-26c.2a), anti-Sca1-APC (D7), anti-IgM-PE (RMM-1), anti-CD16/32-APC/Cy7(93), anti-CD127 (IL-7R)-PerCP/Cy5.5 (SB/199), anti-CD93 (AA4.1)-PE (AA4.1), anti-CD117 (c-kit)-PE/Cy7 (2B8), and anti-CD34-PE (MEC14.7). Anti-human/mouse phospho-ERK1/2 (T202/Y204)-APC (MILAN8R), anti-human-mouse phospho-p38 (T180/Y182)-APC (4NIT4KK), and anti-human/mouse phosphor-AKT (S473)-APC (SDRNR) were purchased from eBioscience (San Diego, CA, USA).

Cells at the third passage (P3) were digested with trypsin to form a single cell suspension solution, which was incubated with antibodies, including anti-CD34-PE, anti-CD31-APC, anti-CD45-PerCP-Cy5-5, anti-CD10-APC-Cy7 (BioLegend, USA), anti-CD13-PE, and anti-CD49d-PE-Cy7 (BD Biosciences, USA). All antibody incubations were performed at 37°C in the dark for 30 min. The cells were analyzed by flow cytometry (LSR II, BD Biosciences, USA) and Treestar FlowJo software.

Purified CD34⁺ cells were evaluated by two-color and three-color flow cytometry after staining in phosphate-buffered saline without calcium chloride or magnesium chloride with the following monoclonal antibodies: anti-CD19-PE, anti-CD34-PE (BioLegend, San Diego, CA, USA), anti-CD38-FITC, anti-CD43-FITC, anti-CD10-FITC (BioLegend), and anti-CD45-PE/Cy5 (BioLegend). Immunofluorescence of the labeled cell membrane was evaluated using a flow cytometer (S3e Cell Sorter; BIO-RAD). Furthermore, phenotype analysis was performed after co-culture of CD34⁺ cells with MS-5^{41 (link)}.

Flow cytometry was used to observe the ADSC phenotypes. Passage 3 ADSCs were trypsinized and incubated with anti-CD34-PE, anti-CD44-PE, anti-CD73-FITC, anti-CD90-FITC, and anti-HLA-DR-PE (Biolegend, San Diego, CA, USA). After three washes with PBS, the fluorescence of ADSCs was observed. To verify adipogenic differentiation and osteogenic differentiation, ADSCs at passage 3 were incubated separately with adipogenic and osteogenic differentiation medium (Gibco, United States) following the supplier’s instructions for 2 or 3 weeks. The induced cells were stained with Oil Red O and Alizarin Red respectively and microscopically imaged.