For cell surface flow cytometry, cells were incubated with human Fc receptor binding inhibitor (No. 14-9161-73, eBioscience, San Diego, CA) followed by primary antibodies CD235-APC (No. 306607, BioLegend, San Diego, CA), CD41-FITC (RRID: AB_314373, BioLegend No. 303703), and CD11b-PE/Cy5 (No. 101209, RRID: AB_312995, BioLegend) or CD-71-APC (No. BD551374, RRID: AB_398500, BD Biosciences, Franklin Lakes, NJ), CD34-Fluor450 (No. 48-0341-82, RRID: AB_468936, eBioscience), CD34-PE/Cy7 (No. 343615, RRID: AB_2629725, BioLegend), and GATA1-PE (No. 13353, RRID: AB_2798187, Cell Signaling Technology, Danvers, MA). Data were collected on a DxP10 flow cytometer (Cytek, Cerritos, CA) and analyzed using FlowJo Software, Version 9.7.2.
Cd71 apc
Manufactured by BD
CD71-APC is a fluorescently-labeled antibody that binds to the transferrin receptor (CD71) on the surface of cells. It can be used to detect and quantify cells expressing CD71 in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Human fc receptor binding inhibitor, by Thermo Fisher Scientific (3 mentions)
Cd11b pe cy5, by BioLegend (3 mentions)
Cd34 fluor450, by Thermo Fisher Scientific (3 mentions)
Cd41 fitc, by BioLegend (3 mentions)
Dxp10 flow cytometer, by Cytek (3 mentions)
Cd235 apc, by BioLegend (3 mentions)
Cd34 pe cy7, by BioLegend (2 mentions)
Gata1 pe, by Cell Signaling Technology (2 mentions)
Cd31 pe, by BD (2 mentions)
Cd34 pe, by BD (2 mentions)
Anti oct4, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Cd45 apc, by BD (1 mentions)
Cd34 fitc, by BD (1 mentions)
Cd235a pe, by BD (1 mentions)
Facsdiva, by BD (1 mentions)
Anti erα, by Santa Cruz Biotechnology (1 mentions)
Cd15 apc, by BD (1 mentions)
Winlist 3d, by Verity Software House (1 mentions)
Ngfr apc, by BD (1 mentions)
8 protocols using cd71 apc
1
Cell Surface Flow Cytometry Analysis
2
Differentiation Marker Analysis of Embryoid Bodies
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EBs were dissociated with Collagenase IV in 37 °C and 5 % CO2 for 2 h. Single cell suspension from dissociated EBs was resuspended in 3 % FBS-PBS. The cell were passed through a 70 μm cell strainer and incubated at 4 °C for 1 h with the following fluorochrome-conjugated mouse anti-human antibodies: CD31-PE, CD34-FITC, CD45-APC, CD235a-PE, and CD71-APC (all BD Biosciences) or their corresponding isotype controls. Anti-OCT4 (BD Biosciences) and anti-ER-α (Santa Cruz) staining was identified using Alexa 488- and 647-conjugated goat anti-mouse IgG (Invitrogen). After washing with 3 % FBS-PBS, the cells were stained with 7-amino actinomycin to exclude dead cells. Flow analysis was performed on a FACSCanto II running BD FACSDiva™ (BD Biosciences) and acquired data were analyzed using FlowJo version 10 (Tree Star, Inc.).
3
FACS Analysis of Cell Populations
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All fluorescence-activated cell sorter (FACS) analyses were performed on a FACScalibur (Becton-Dickinson [BD], Alpen a/d Rijn, the Netherlands) and data were analyzed using WinList 3D (Verity Software House, Topsham, USA). Cells were sorted on a MoFlo sorter. Antibodies: CD34-PE, NGFR-APC, CD14-PE, CD15-APC, CD71-APC and GPA-PE were obtained from BD. Viability was assessed using Annexin V APC (IQ Products, Groningen, The Netherlands) according to the manufacturer's recommendations. Briefly, cells were harvested, resuspended in 100 µL calcium buffer containing 5 µL Annexin V, and incubated for 20 min at 4°C in the dark, washed with 5 mL calcium buffer and binding of APC-conjugated Annexin V was measured by FACS.
4
Cell Surface Flow Cytometry Analysis
5
Erythroid Progenitor Cell Differentiation
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BM- and CB-derived erythroid progenitor cells were differentiated using the three phase EDM. The differentiation state of the cells was determined by staining the cells with fluorescently conjugated antibodies specific to erythroid surface markers: CD36-FITC (BD Biosciences, 20 µL/test), CD71-APC (BD Biosciences, 20 µL/test) and CD235a-BV421 (BD Biosciences, 5 µL/test). The antibody co-stained cells were subjected to FACS (Sony SH800, Sony Biotechnology) and were gated based on differential expression of CD36, CD71, and CD235a on different days of erythroid differentiation: populations 1 and 2 (day 0), populations 3 and 4 (day 4), populations 5, 6, and 7 (day 10 for BM and day 11 for CB). Populations 6 and 7 for BM and CB cells were collected on different days of differentiation to account for slight differences in speed of maturation of BM and CB cells. Purity of the sorted cell populations was assessed by flow cytometry, centrifugation, and cytospin analyses.
6
Isolation and Analysis of CD34+ Cells
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Transduced CB CD34+ cells were sorted for green fluorescence (GFP) and CD34 expression after staining with an APC-conjugated anti-CD34 antibody (BD340667, Becton Dickinson), using a fluorescence-activated cell sorting (FACS) Vantage cell sorter. Cells were stained with the following antibodies: CD34-APC (BD340667, BD), CD34-PE (BD348057, BD), CD71-APC (BD561940, BD), Glycophorin A-PE (MHGLA04, Invitrogen), CD31-PE (BD555446, BD), CD34-PEcy7 (BD560710, BD), and CD45-APC (MHCD4505, Invitrogen), and analyzed by FACS. Neural progenitors were stained with Annexin V (BD556421, BD) and Ki-67 (BD558615, BD) antibodies upon fixation. Isotype-matched antibodies were utilized as negative controls.
7
Cell Surface Marker Analysis by Flow Cytometry
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For cell surface flow cytometry, cells were incubated with human Fc receptor binding inhibitor (#14-9161−73; eBioscience) followed by primary antibodies CD235−APC (#306607; BioLegend) CD41-FITC (#303703; BioLegend) and CD11b-PE/Cy5 (#101209; BioLegend) or CD-71-APC (BD551374; BD Biosciences), p53-PECy7 (NB200-171; Novus), Sca-1-PE (#12-5981-81; eBioscience), CD34-Fluor®450 (#48-0341-82; eBioscience) and CD16/32-FITC (#101305; BioLegend). FRET measurements were obtained as described69 (link). To measure ECFP and FRET cells were excited with the 405 nm laser and fluorescence was collected in the ECFP channel with a standard 450/40 filter, while the FRET-signal was measured with a 529/24 filter. To measure EYFP, cells were excited with the 488 nm laser while emission was also taken with a 529/24 filter. Data were collected on a DxP10 flow cytometer (Cytek) and analyzed by using FlowJo Software, v.9.7.2.
8
Hematopoietic Lineage Analysis Protocol
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Hematopoietic cell differentiation was performed as previously described (Ronn et al., 2015 , Woods et al., 2011 (link)). Micrographs of cells from the cultures were taken at day 22 prior to whole well harvest. Floating and individualized cells were pooled, washed, and divided into two samples. One sample was used for assessment of hematopoietic lineage markers using FACS. The other sample was plated into methylcellulose (MethoCult H4435, StemCell Technologies) for colony-forming unit assay. The cells for FACS analysis were stained for mouse anti-human CD45/CD43/CD34/CD71 antibodies (BD Pharmingen; CD71APC, cat. no. 551374, clone M-A712. CD45 FITC or PE cat. no. 555482, 555483, clone HI30. CD43FITC cat. no. 555475, clone 1G10. CD34APC cat. no. 555824, clone 581), CD33/CD235a (GPA) antibodies (eBioscience, CD235a(Gly-A) cat. no. 12-9987-82, clone HIR2(GA-R2). CD33PE-Cy7 cat. no. 25-0338-42 clone WM-53(WM53)) and analyzed by a FACSCanto II flow cytometer.
Microarray data were evaluated at the level of gene sets to define and quantitate trends in gene expression similar to published data. Ranked gene lists were created and submitted to the online public repository provided by the BROAD Institute for Gene Set Enrichment Analysis (GSEATo) (Subramanian et al., 2005 (link)). Venn diagram analysis was performed using Microsoft PowerPoint tool software.
Microarray data were evaluated at the level of gene sets to define and quantitate trends in gene expression similar to published data. Ranked gene lists were created and submitted to the online public repository provided by the BROAD Institute for Gene Set Enrichment Analysis (GSEATo) (Subramanian et al., 2005 (link)). Venn diagram analysis was performed using Microsoft PowerPoint tool software.
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