The lentivirus vector PLV-H1-EF1α-period was derived from Biosettia (USA). Fetal bovine serum (FBS), trypsin, penicillin/streptomycin, OPTI-MEM, sodium pyruvate, non-essential amino acids (NEAA), and l -glutamine were purchased from Gibco (USA). The SYBR Green PCR kit was acquired from TaKaRa Biotech (China). The EcoR I and Xba I restriction endonucleases were obtained from New England BioLabs (UK). DMEM and RPMI 1640 were purchased from Neuronbc (China). The H4434 was acquired from Stem Cell Technologies (Canada). Annexing-V/PI kit and the antibodies against human CD11b (PE), CD14 (APC), and CD71 (PE) were purchased from BioLegend (USA).
Cd71 pe
Manufactured by BioLegend
Sourced in United States
CD71-PE is a fluorescently-labeled antibody that specifically binds to the CD71 (Transferrin Receptor) antigen on the surface of cells. It can be used for the identification and analysis of CD71-expressing cells by flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Epidermal growth factor (egf), by Thermo Fisher Scientific (2 mentions)
Cd49f apc, by Thermo Fisher Scientific (2 mentions)
Ham s f 12, by Merck Group (2 mentions)
Cholera toxin, by Merck Group (2 mentions)
Facsdiva, by BD (2 mentions)
Cacl2, by Merck Group (2 mentions)
Facsaria 3 cell sorter, by BD (2 mentions)
Ter119 fitc, by BioLegend (2 mentions)
Cd11b bv510, by BioLegend (2 mentions)
Murine trustain fcx, by BioLegend (2 mentions)
Alexafluor700 f4 80, by Bio-Rad (2 mentions)
Percp cy5.5 cd11c, by BD (2 mentions)
Siglecf pe cf594, by BD (2 mentions)
Cd54 apc, by BioLegend (2 mentions)
Pb cd40, by BioLegend (2 mentions)
Bv711 cd64, by BioLegend (2 mentions)
Bv605 ia ie, by BD (2 mentions)
Buv737 cd43, by BD (2 mentions)
Gr1 apc cy7, by BioLegend (2 mentions)
Cd206 pe cy7, by BioLegend (2 mentions)
12 protocols using cd71 pe
1
Lentiviral Vector-Mediated Gene Delivery
2
Multiparametric Flow Cytometry Staining
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Staining for flow cytometry analysis was performed using cryopreserved PBMCs. Cells were stained for 30 min on ice with biotinylated recombinant HAs diluted in in 2% FBS and 2 mM EDTA in PBS (P2), washed twice, then stained for 30 min on ice with Alexa 647-conjugated S, IgA-FITC (M24A, Millipore, 1:500), IgG-BV480 (goat polyclonal, Jackson ImmunoResearch, 1:100), IgD-SB702 (IA6–2, Thermo, 1:50), CD38-BB700 (HIT2, BD Horizon, 1:500), CD20-Pacific Blue (2H7, 1:400), CD4-BV570 (OKT4, 1:50), CD24-BV605 (ML5, 1:100), streptavidin-BV650, CD19-BV750 (HIB19, 1:100), CD71-PE (CY1G4, 1:400), CXCR5-PE-Dazzle 594 (J252D4, 1:50), CD27-PE-Cy7 (O323, 1:200), IgM-APC-Fire750 (MHM-88, 1:100), CD3-APC-Fire810 (SK7, 1:500), and Zombie NIR (all BioLegend) diluted in Brilliant Staining buffer (BD Horizon). Cells were washed twice with P2 and acquired on an Aurora using SpectroFlo v2.2 (Cytek). Flow cytometry data were analyzed using FlowJo v10 (Treestar). In each experiment, PBMC were included from convalescent and control participants.
3
Multiparametric Phenotyping of Mouse Cells
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The following directly labeled anti-mouse monoclonal antibodies were used for BM cell phenotypical analysis: CD90.2-APC and CD45-PE/Cy7 for lymphoid progenitors, CD61-APC and CD41-FITC for megakaryocytic population, CD71-PE and Ter119-FITC for erythroid precursors, CD11b-PE and Gr1-FITC for granulocytes/monocytes progenitors, Lineage Cocktail (CD3, Gr1, CD11b, CD45R, Ter119)-FITC, Sca1-PE, cKit (CD117)-APC for hematopoietic stem cells, all purchased from BioLegend (BioLegend, San Diego, CA, USA).
The phenotypical analysis of splenocytes was performed using the following anti-mouse antibodies: CD4-PE/Cy5, CD8a-PE (BioLegend) for helper and cytotoxic T cells, CD19 (BioLegend) for B cells, CD11c-PE, I-Ab-FITC, and TLR4 (CD284)-PE/Cy7 (all from BioLegend) for dendritic cells (DCs), and NK1.1-FITC (BioLegend) for NK cells. To detect proliferative cells, Ki67-eFluor660 (eBioscience, San Diego. USA) was used.
Single-cell suspensions of splenocytes or BM cells were incubated with the fluorescently labeled antibodies in PBS containing 1% BSA, at 4°C for 20 min for cell surface staining. For intracellular staining (Ki67), cells were permeabilized using the Foxp3 Fix/Perm Buffer (eBioscience), according to the manufacturer’s instructions. Measurements were performed with a FACSCalibur flow cytometer as described above.
The phenotypical analysis of splenocytes was performed using the following anti-mouse antibodies: CD4-PE/Cy5, CD8a-PE (BioLegend) for helper and cytotoxic T cells, CD19 (BioLegend) for B cells, CD11c-PE, I-Ab-FITC, and TLR4 (CD284)-PE/Cy7 (all from BioLegend) for dendritic cells (DCs), and NK1.1-FITC (BioLegend) for NK cells. To detect proliferative cells, Ki67-eFluor660 (eBioscience, San Diego. USA) was used.
Single-cell suspensions of splenocytes or BM cells were incubated with the fluorescently labeled antibodies in PBS containing 1% BSA, at 4°C for 20 min for cell surface staining. For intracellular staining (Ki67), cells were permeabilized using the Foxp3 Fix/Perm Buffer (eBioscience), according to the manufacturer’s instructions. Measurements were performed with a FACSCalibur flow cytometer as described above.
4
Cell Surface Marker Profiling
5
Primary KC Isolation and Culture
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A suspension of primary KCs with 5.0 × 106cells/ml was incubated with CD71-PE (1:200, BioLegend) and CD49f-APC (1:85, eBioscience). The selection of α6bri/CD71dim cells was performed using a FACSAriaIII Cell Sorter and FACSDiva software (BD Biosciences). Sorted α6bri/CD71dim cells were plated at 1.8 × 103 cells/cm2 onto the feeder layer and cultured in FAD medium [1-part Ham’s F-12 (Sigma-Aldrich) and 3-parts DMEM supplemented with 10% non-inactivated FBS, 1.8 × 104 M adenine, 0.5 μg/ml hydrocortisone, 5 μg/ml insulin, 10–10 M cholera toxin (Sigma-Aldrich), 10 ng/ml epidermal growth factor (Peprotech), 1.8 mM CaCl2 (Merck), and 1% PenStrep].
6
Multicolor Flow Cytometry of PBMC
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We isolated peripheral blood mononuclear cells (PBMC) from peripheral blood by Ficoll gradient and we stored them in liquid nitrogen for batched analysis. For multicolor flow cytometry analyses, we used the following monoclonal antibodies: from Becton Dickinson (BD, Franklin Lakes, NJ), CD3-FITC, CD3-PerCP-Cy5.5, CD4-APC-Cy7, CD8-BV510, CD45RO-FITC, CD45RA-APC, CD45RA-APC-Cy7, CD71-PE, IgD-PerCP-Cy5.5, CD25-APC-Cy7, CD138-BV421, CCR4-PE, CD27-PE, CD95-BV421, CD28-BV421, TIM3-BV421, CCR6-BV421, CCR7-AF700, CXCR3-PE, IL-2-PE, IL-17-BV786, and IFN-γ-PE-Cy7; from Biolegend (San Diego, CA), CD19-BV510, CD56-FITC, CD27-APC, CD127-FITC, CD57-PerCp-Cy5.5, PD-1-APC-Cy7, CXCR5-FITC, LAG3-APC, IL-10-PerCP-Cy5.5, and TNF-α-FITC. From eBioscience (San Diego, CA), CD4-PE-Cy7, CD21-PE, and CD24-APC-Cy7 were used. From Miltenyi Biotec (Auburn, CA), CD25-APC and anti-KLRG1-PE were used. From Beckman Coulter (Indianapolis, IN), CD38-PE-Cy7 was used.
We performed intracellular staining for IL-2, IL-4, IL-17, IFN-g, and TNF-a together with extracellular markers for CD4+, CD8+, and CD19+. Cells were fixed and permeabilized using Intracellular Fixation and Permeabilization Buffer Set (eBioscience) according to the manufacturer’s instructions.
Data were acquired (> 1 × 106 events) on a 3-laser FACSLyric flow cytometer (BD Biosciences) and analyzed with FlowJo® software.
We performed intracellular staining for IL-2, IL-4, IL-17, IFN-g, and TNF-a together with extracellular markers for CD4+, CD8+, and CD19+. Cells were fixed and permeabilized using Intracellular Fixation and Permeabilization Buffer Set (eBioscience) according to the manufacturer’s instructions.
Data were acquired (> 1 × 106 events) on a 3-laser FACSLyric flow cytometer (BD Biosciences) and analyzed with FlowJo® software.
7
Erythroid Lineage Analysis in Human Bone Marrow
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Human bone marrow was stained with a lineage cocktail using FITC-conjugated antibodies to CD2, CD7, CD11b and CD14 and erythroid markers CD71-PE (334105, BioLegend), CD326-APC (324207, BioLegend) and CD235a-BV421 (349131, BioLegend) and DAPI to identify viable cells. Cell sorting was performed on a FACSAria II to collect four populations (CD71+CD326+CD235a–, CD71+CD326+CD235a+, CD71+CD326–CD235a+ and CD71–CD326–CD235a+) or sorted on an MA900 where cells were stained with Percp/Cy5.5-conjugated antibodies to CD2, CD7, CD11b and CD14 and erythroid markers CD71-FITC (334103, BioLegend), CD326-APC (BioLegend, clone 9C4) and CD235a-BV421 (BioLegend, clone HI264).
8
Primary KC Isolation and Culture
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A suspension of primary KCs with 5.0 × 106cells/ml was incubated with CD71-PE (1:200, BioLegend) and CD49f-APC (1:85, eBioscience). The selection of α6bri/CD71dim cells was performed using a FACSAriaIII Cell Sorter and FACSDiva software (BD Biosciences). Sorted α6bri/CD71dim cells were plated at 1.8 × 103 cells/cm2 onto the feeder layer and cultured in FAD medium [1-part Ham’s F-12 (Sigma-Aldrich) and 3-parts DMEM supplemented with 10% non-inactivated FBS, 1.8 × 104 M adenine, 0.5 μg/ml hydrocortisone, 5 μg/ml insulin, 10–10 M cholera toxin (Sigma-Aldrich), 10 ng/ml epidermal growth factor (Peprotech), 1.8 mM CaCl2 (Merck), and 1% PenStrep].
9
Erythroid Progenitor Analysis in Mouse Bone Marrow
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For the analysis of erythroid progenitors in BM, CD71/TER119 double labeling was performed as described [13 (link),14 (link)]. BM cells were isolated from femurs of 18–22 M-old mice and washed with 5% fetal bovine serum-containing Dulbecco’s phosphate buffered saline without calcium and magnesium (5% FBS-PBS). After blocking the Fc receptor using anti-mouse CD16/32 antibodies (Biolegend, San Diego, CA), BM cells were incubated on ice for 1 h with FITC-Ter119 (Biolegend) and PE-CD71 (Biolegend) after blocking Fc receptors. Control samples were incubated with the FITC-IgG2b and PE-IgG1 isotype control antibodies (Biolegend). 7AAD dye (COSMO BIO, Tokyo, Japan) was added 5 min before flow cytometry analysis to exclude dead cells. Flow cytometry was performed using the BD LSRFortessa X-20 cell analyzer (BD Biosciences). All Ter119-positive cells were classified into four subsets: ProE (Ter119medCD71high), EryA (Ter119highCD71highFSChigh), EryB (Ter119highCD71highFSClow), and EryC (Ter119highCD71lowFSClow), corresponding to morphologically recognized proerythroblasts and basophilic, polychromatic, and orthochromatic erythroblasts, respectively.
10
Fetal Liver Cell Immunophenotyping
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E14.5 fetal liver cells were dissociated and resuspended in PBS with 2% FBS and passed through 25 μm cell strainers to obtain single-cell suspensions prior to antibody staining. Erythroid and myeloid populations were analyzed using combinations of APC-Ter-119, PE Cy7-c-Kit (Biolegend, 105814), PE-CD71 (Biolegend, 113808), Mac1-APCe780 (47-0112-82) and/or Gr1-PE-Cy7 (Biolegend, 108416) at 4°C for 30 min. After staining, cells were washed once with 2% FBS, 10 mM glucose and 2.5 mM EDTA in PBS. The cells were analyzed on an Attune™ NxT Flow Cytometer (Thermofisher Scientific) or collected on a FACSAria II cell sorter (BD Biosciences). To quantify apoptosis and CD71/Ter119 levels, the cells were washed in Annexin V Buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) and stained with Annexin V-Pacific blue (ThermoFisher, A35122) (1:40) and DRAQ7 (Abcam, ab109202) (1:100) for 20 min in the dark at room temperature. After staining, cells were washed once in PBS containing 10% FBS or PBS containing 2% FBS, 10 mM glucose and 2.5 mM EDTA. The stained cells were analyzed on a Attune™ NxT Flow Cytometer flow cytometer (Thermofisher Scientific) or collected on a FACSAria II cell sorter (BD Bioscience). The data were analyzed using FlowJo v10.1 software (TreeStar).
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