For ex vivo surface staining, 100 μl of blood was immediately diluted in equal parts with DPBS. After centrifugation, the cell pellet was stained with an antibody mixture containing the following antibodies: For identification of T helper cells, we included CD4-BV510 (OKT4, Biolegend) and CD8-BV785 (RPA-T8, Biolegend). For identification of naïve, central and effector memory T-cells, we used CD45RA-FITC (HI100, Biolegend) and CCR7-PE-Cy7 (3D12, BD). In addition, the γc cytokine receptors IL-7Rα AF700 (clone A019D5, Biolegend), IL-2Rα-PerCP/Cy5.5 (BC96, Biolegend), IL-2Rβ (CD122)-PE (TU27, Biolegend), IL-15Rα (CD215)-APC (JM7A4, Biolegend), and γc (CD132)-PE-CF594 (TUGh4, BD) were included. Fixable viability dye eFluor780 (Thermo Fisher Scientific) was used to exclude dead cells. Staining was performed in triplicates. Sample measurement was performed on a LSR Fortessa flow cytometer (BD Biosciences). For data analysis FlowJo software (Miltenyi Biotech) was used. The gating procedure is depicted as Supplementary Figures 1A,B . Dead cells (viability marker positive cells) were excluded.
Cd45ra fitc hi100
Manufactured by BioLegend
CD45RA-FITC (HI100) is a fluorescently labeled antibody that binds to the CD45RA protein, which is expressed on the surface of certain types of immune cells. This product is intended for use in flow cytometry applications to identify and analyze these cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Ccr7 pecy7 3d12, by BD (1 mentions)
Fixable viability dye efluor 780, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
7 aminoactinomycin d, by Thermo Fisher Scientific (1 mentions)
Easysep human cord blood cd34 positive selection kit 2, by STEMCELL (1 mentions)
Cd38 pe cy7 hb 7, by BioLegend (1 mentions)
Cd90 pe 5e10, by BD (1 mentions)
Facsaria 3, by BD (1 mentions)
2 protocols using cd45ra fitc hi100
1
Ex vivo surface staining of T cell markers
2
Isolation and Characterization of Hematopoietic Stem and Progenitor Cells from Umbilical Cord Blood
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Anonymized human UCB samples were collected from healthy new-borns of both sexes at University Hospital Basel. Relevant ethical regulations were followed, according to the guidelines of the local Basel ethics committees (vote 13/2007V, S-112/2010, EKNZ2015/335). UCB cells were processed by density gradient centrifugation, and CD34+ cells were isolated using EasySep™ human cord blood CD34 positive selection kit II (cat# 17896, Stemcell Technologies, Vancouver, BC, Canada) and stored at -150 °C. Upon thawing, CD34+ cells were stained with the following antibodies: CD34 (APC conjugate, clone: 581, 1 : 100 dilution, BD Biosciences), CD38 (PEcy7, HB7, 1 : 200, BioLegend), CD45RA (FITC, HI100, 1 : 100, BioLegend), CD90 (PE, 5E10, 1 : 50, BD Biosciences) and CD49f (PEcy5, GoH3, 1 : 50, BD Biosciences) for 30 minutes at 4 °C. Cells were then washed and resuspended in PBS + 2% FCS with 1 μg ml -1 7-aminoactinomycin D (7-AAD, ThermoFisher Scientific). HSPCs, phenotypically defined as CD34+CD38-CD45RA -CD90+CD49f+, were sorted using a BD™ FACSAria III with 100 μm nozzle, purity mode and sorting purities ≥90%. Gates and thresholds were set according to fluorescence minus one (FMO) controls.
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