Flow cytometry was implemented for measuring UC-MSC surface markers. Briefly, cells were incubated with FITC-conjugated primary antibodies after preprocessing steps as described in the section of cell cycle analysis. Then, the cells were subjected to a MACSQuant Analyzer Flow Cytometer (Miltenyi Biotec) for analysis. The primary antibodies used in this experiment are mouse anti-human CD34 (555821; BD Pharmingen), mouse anti-human CD45 (555482; BD Pharmingen), mouse anti-human CD73 (561254; BD Pharmingen), mouse anti-human CD90 (561969; BD Pharmingen), mouse anti-human CD105 (561443; BD Pharmingen), and mouse anti-human HLA-DR (555560; BD Pharmingen).
Mouse anti human cd34
Manufactured by BD
Sourced in United States
Mouse anti-human CD34 is a monoclonal antibody that recognizes the CD34 antigen, a cell surface glycoprotein expressed on hematopoietic stem and progenitor cells. It is commonly used in flow cytometry and immunohistochemistry applications to identify and enumerate CD34-positive cells.
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Lab products found in correlation
Mouse anti human cd45, by BD (4 mentions)
Mouse anti human cd90, by BD (4 mentions)
Mouse anti human cd73, by BD (3 mentions)
Mouse anti human cd105, by BD (3 mentions)
Mouse anti human cd44, by BD (3 mentions)
Mouse anti human hla dr, by BD (2 mentions)
Rat anti human cd49f, by BD (2 mentions)
Mouse anti human cd38, by BD (2 mentions)
Mouse anti human cd45ra, by BD (2 mentions)
Macsquant analyzer flow cytometer, by Miltenyi Biotec (1 mentions)
Sorafenib, by Selleck Chemicals (1 mentions)
7 aminoactinomycin d, by Merck Group (1 mentions)
Rat anti mouse cd45, by BD (1 mentions)
Annexin 5 cy5, by BD (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Flow tube, by Corning (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Ab131260, by Abcam (1 mentions)
Lsrfortessa, by BD (1 mentions)
7 protocols using mouse anti human cd34
1
Comprehensive Flow Cytometry Analysis of UC-MSC
2
Evaluation of Hypoxia-Targeting Agents
3
Characterizing hiPSC-derived MSCs and rat BMSCs
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The hiPSC-derived MSCs or rat BMSCs were harvested using 0.25% trypsin/ethylenediaminetetraacetic acid and resuspended in 100 μl staining medium (2% FBS and 2% N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid in PBS). The cells were stained with mouse anti-human CD29 (BD, USA), mouse anti-human CD44 (BD, USA), mouse anti-human CD34 (BD, USA), mouse anti-human CD105 (BD, USA), and mouse anti-human CD45 (BD, USA) antibodies for 30 min at 4℃. Isotype-matched antibody (immuno-globulin G 2b-fluorescein isothiocyanate) was used to determine nonspecific fluorescence. Samples were run on a cytometer (CytoFLEX Beckman Coulter, USA).
4
Isolation and Characterization of Urine-Derived Stem Cells
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Primary culture of the USCs was performed as previously described [33 (link),34 (link)]. Briefly, the USCs were obtained from four young male adult donors (20–30 years old). Fresh urine sample (about 200 mL) was centrifuged and washed with PBS for twice. The sediment was re-suspended and cultured in T25 cell culture flasks with 5 mL of established medium [35 (link)].
Cells in logarithmic growth phase were digested with trypsin (Gibco, USA), with their density adjusted to 1 × 106 cells/mL with PBS. 100 μL aliquot of cell suspension was moved to each flow tube (Corning, USA). Thereafter, 2 μL of mouse anti-human CD29 (559883, BD Pharmingen™, USA), mouse anti-human CD34 (555821, BD Pharmingen™), mouse anti-human CD44 (555478, BD Pharmingen™), mouse anti-human CD73 (550257, BD Pharmingen™), mouse anti-human CD90 (561558, BD Pharmingen™) and mouse anti-human CD133(130-111-085, Miltenyi Biotec GmbH, Germany) antibodies were added to each tube. The mixture was allowed to incubate for 30 min in darkness at room temperature. Thereafter, the cells were washed twice with PBS and re-suspended in 200 μL PBS for flow analysis.
Cells in logarithmic growth phase were digested with trypsin (Gibco, USA), with their density adjusted to 1 × 106 cells/mL with PBS. 100 μL aliquot of cell suspension was moved to each flow tube (Corning, USA). Thereafter, 2 μL of mouse anti-human CD29 (559883, BD Pharmingen™, USA), mouse anti-human CD34 (555821, BD Pharmingen™), mouse anti-human CD44 (555478, BD Pharmingen™), mouse anti-human CD73 (550257, BD Pharmingen™), mouse anti-human CD90 (561558, BD Pharmingen™) and mouse anti-human CD133(130-111-085, Miltenyi Biotec GmbH, Germany) antibodies were added to each tube. The mixture was allowed to incubate for 30 min in darkness at room temperature. Thereafter, the cells were washed twice with PBS and re-suspended in 200 μL PBS for flow analysis.
5
Multiparametric Flow Cytometry Analysis of Immune Cell Populations
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Cells were washed with ice-cold phosphate-buffered saline (PBS) and stained with corresponding antibodies in PBS containing 2% fetal bovine serum and 0.02% sodium azide. We used: mouse anti-human CD38 (BD, #563964), mouse anti-human CD34 (BD, #348811), rat anti-human CD49f (BD, #563271), mouse anti-human CD90 (BD, #562685), and mouse anti-human CD45RA (BD, #560673). For intracellular NE protein analysis, cells were subsequently fixed and permeabilized using the Fix&Perm kit (Nordic Mubio, #GAS-002FOC), followed by incubation with a rabbit anti-human NE (abcam, #ab131260) antibody for 15 minutes (min) at room temperature, and subsequent incubation with a goat polyclonal secondary antibody to rabbit IgG-H&L (Alexa Fluor 488) (abcam, #ab150077) for 15 min at room temperature. Cells were fixed with 0.5% paraformaldehyde and measured using a BD LSRFortessa.
Additional methods are available in the Online Supplementary Appendix.
Additional methods are available in the Online Supplementary Appendix.
6
Multiparametric Flow Cytometry Analysis of Stem Cell Markers
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Fourth‐passage cells (5 × 105) of each group were harvested and fixed with 4% paraformaldehyde for 30 minutes, followed by incubation with mouse anti‐human p75NTR, mouse anti‐human CD44, mouse anti‐human CD73, mouse anti‐human CD90, mouse anti‐human CD105, mouse anti‐human CD11b, mouse anti‐human CD19, mouse anti‐human CD34, mouse anti‐human CD45 and mouse anti‐human HLA‐DR antibodies (1:20; BD Pharmingen™) at 4°C for 2 hours. Subsequently, the specimens were analysed by flow cytometry.
7
Characterizing Hematopoietic Stem Cells
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BM mononuclear cells were washed with phosphate-buffered saline (PBS) and stained with corresponding surface marker antibodies in PBS containing 2% fetal bovine serum (FBS) and 0.02% sodium azide. For surface marker analysis (panel adapted from van Galen et al. 21 ), the following antibodies were used: mouse anti-human CD38 (BD, #563964), mouse anti-human CD34 (BD, #348811), rat anti-human CD49f (BD, #563271), mouse antihuman CD90 (BD, #562685), mouse anti-human CD45RA (BD, #560673), mouse anti-human CD10 (BD, #563734), and mouse anti-human CD135 (BD, #564708). For the analysis of intracellular gH2AX protein, cells were subsequently permeabilized and fixed using the IntraSure TM Kit (BD #641776), followed by incubation with mouse anti-human gamma gH2AX (BD, #560445) antibody. For the analysis of ROS, cells were incubated with 20 μM 2ʹ,7ʹ-dichlorofluorescin diacetate (DCFH-DA) (Sigma-Aldrich/Merck, #D6883) for 10 min at 37 °C after staining with surface marker antibodies.
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