Single cells were directly sorted into 96-well plates with erythroid/megakaryocyte bipotential medium for erythroid and megakaryocytic output (Psaila et al., 2016 (link)). StemSpan (StemCell Technologies, Inc.) was supplemented with thrombopoietin (TPO; 100 ng/ml), erythropoietin (EPO; 1 IU/ml), stem cell factor (SCF; 100 ng/ml), IL-3 (10 ng/ml), IL-6 (10 ng/ml; all from Peprotech), and human low-density lipoprotein (40 mg/ml; StemCell Technologies, Inc.). Single cells were cultured at 37°C in 5% CO2 for 2 wk, and colony readout was performed by direct light microscopy and flow cytometry using CD41a APC (eBioscience; Clone: HIP8, Cat# 17–0419-42, RRID: AB_2573144), CD42b PE (eBioscience; Clone: HIP1, Cat# 12–0429042, RRID: AB_10852864), CD11b FITC (eBioscience; Clone: ICRF44, Cat# 11–0118-42, RRID: AB_1582242), CD14 FITC (eBioscience; Clone: 61D3, Cat# 11–0149-41, RRID: AB_10597445), CD34 APC–eFluor 780 (Clone: 4H11, Cat# 47–0349-42, RRID: AB_2573956), and CD235a PerCp Cy5.5 (Biolegend; Clone: HIR2, Cat# 306614, RRID: AB_10683170). CD41a and CD42b double-positive cells were used to identify the megakaryocytic output.
Cd235a percp cy5
Manufactured by BioLegend
Sourced in United States
CD235a PerCp Cy5.5 is a fluorochrome-conjugated antibody that binds to the CD235a (Glycophorin A) antigen. CD235a is a sialoglycoprotein found on the surface of red blood cells and is commonly used as a marker for erythroid lineage cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b fitc, by Thermo Fisher Scientific (1 mentions)
Cd14 fitc, by Thermo Fisher Scientific (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (1 mentions)
Human low density lipoprotein, by STEMCELL (1 mentions)
Stemspan, by STEMCELL (1 mentions)
Cd34 pe cy7, by BioLegend (1 mentions)
Lsrfortessa sorp, by BD (1 mentions)
High throughput sampler, by BD (1 mentions)
Cd45ra bv421, by BioLegend (1 mentions)
Cd14 bv605, by BioLegend (1 mentions)
Cd90 pe cy5, by BioLegend (1 mentions)
Cd10 pe dazzle594, by BioLegend (1 mentions)
Cd41a apch7, by BD (1 mentions)
2 protocols using cd235a percp cy5
1
Single-cell bipotent erythroid-megakaryocyte culture
2
Hematopoietic Subset Analysis by Flow
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The flow cytometry panel used to define hematopoietic subsets was composed of CD34-PECy7, CD90-PECY5, CD38-AF488, CD371-PE, CD45Ra-BV421, CD14-BV605, CD42b-AF647, CD10-PEDazzle594, CD235a-PerCPCy5.5 (BioLegend, California), CD15-BUV395, CD41a-APCH7, CD71-BV786 (BD Biosciences, California), and DAPI for dead cell exclusion (ThermoFisher, California). In short, Day 6 cells were harvested, FcR-blocked (FcR binding inhibitor, ThermoFisher), surface-stained, then analyzed in a buffer containing TruCount Control Beads using a BD LSR Fortessa SORP fitted with a high-throughput sampler (BD Biosciences, California). Data was analyzed in Diva 6.0.2 and exported to CSV where after all cell data was transformed to cell per mL then normalized on a per-donor basis to untreated control wells to determine drug impact.
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