For flow cytometry sample preparation, the cell samples were harvested and collected in EasySep buffer (StemCell Technologies) at a concentration of 10 × 106 cells/ml. The samples were incubated for 20 min in the dark on ice with antibodies Alexa fluor 647 mouse anti-human TRA-1-60 (BD Biosciences), FITC mouse anti-human TRA-1-81 (BD Biosciences), PE mouse anti-human CD140b (BD Biosciences), and APC-Cy7 mouse anti-human CD31 (BD Biosciences). Next, the cell samples were diluted ten times in EasySep buffer, and centrifuged [350 × g × 5 min]. Supernatant was removed and the dilution step was repeated. After supernatant was removed, the cell samples were resuspended in EasySep buffer at a concentration of 3.33 × 106 cells/ml and filtered through a round-bottom tube with cell strainer (Falcon). The stained cell samples were analyzed using the BD FACSAria™IIu machine and the accompanied BD FACSDiva software. Acquisition of 10,000 events was made, and data analysis was performed after gating for single cell population minus cell debris.
Pe mouse anti human cd140b
Manufactured by BD
The PE mouse anti-human CD140b is a flow cytometry reagent that binds to the CD140b cell surface antigen expressed on various cell types. It can be used to identify and quantify CD140b-positive cells in samples.
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Lab products found in correlation
Facsdiva software, by BD (1 mentions)
Cell strainer, by BD (1 mentions)
Facsaria iiu machine, by BD (1 mentions)
Easysep buffer, by STEMCELL (1 mentions)
Isotype control, by Thermo Fisher Scientific (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Pe mouse anti human cd31, by BioLegend (1 mentions)
2 protocols using pe mouse anti human cd140b
1
Flow Cytometry Sample Preparation
2
Phenotyping Microvascular Endothelial Cells
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Flow cytometric analysis was conducted to detect the major cell populations presented in iMVs-VCs. Microvessels were dissociated into single cells according to the above method. The cell mixture was washed and resuspended in 2%FBS/PBS containing the following antibodies for 30 min at 4°C: Isotype control (Thermo, PA5-33198), PE Mouse anti-Human CD31 (Biolegend, 303105, 5 μL/test), and PE Mouse anti-Human CD140b (BD, 558821, 5 μL/test). Then, cells were washed three times and resuspended in 2%FBS/PBS for analysis by an Accuri C6 flow cytometer (BD). The control group was used to exclude debris and doublets and yield the total cell population. Gating the negative cells was critical in this assay. Subsequently, CD31-positive and CD140b-positive cells were gated according to the Isotype control. The data were analyzed using FlowJo V10 software.
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