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Cd71 fitc

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CD71-FITC is a fluorochrome-conjugated antibody that binds to the human transferrin receptor (CD71). It is used for the detection and analysis of cells expressing CD71, which is a protein involved in the cellular uptake of iron. The CD71-FITC antibody is commonly used in flow cytometry applications to study the expression of CD71 on various cell types.

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Lab products found in correlation

Cd11b pe, by Thermo Fisher Scientific (2 mentions) Red blood cell lysis buffer, by Biosharp (1 mentions) Cd69 fitc, by Thermo Fisher Scientific (1 mentions) Cytoflex s, by Beckman Coulter (1 mentions) Cd49d apc, by BD (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Azide, by Merck Group (1 mentions) Facscanto 2, by BD (1 mentions) Facsdiva, by BD (1 mentions) Annexin 5 fitc, by BD (1 mentions) C kit pe, by Thermo Fisher Scientific (1 mentions) Draq5, by Thermo Fisher Scientific (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Cd45 pe, by BD (1 mentions) Cd235a pe, by BioLegend (1 mentions) Analysis software, by FlowJo (1 mentions) Cd45 v450, by BD (1 mentions) Cd34 pe, by Thermo Fisher Scientific (1 mentions) Cd43 apc, by Thermo Fisher Scientific (1 mentions) Cd41a fitc, by BD (1 mentions)

9 protocols using cd71 fitc

1

Immune Profiling of Ophthalmic Blood

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First, 20 μL of peripheral blood was collected from the ophthalmic vein and added into tubes containing 4 μL of 1% heparin sodium. Red blood cell lysis buffer was added (Biosharp, China) and the blood was lysed in the dark for 2 min. Then, 2 mL of PBS was added to wash off the lysate. Next, the cells were redissolved in PBS and stained in the dark for 30 min with CD206-phycoerythrin (PE), CD11b-PE, CD25-Fluorescein isothiocyanate (FITC), CD69-FITC, and CD71-FITC (eBioscience, USA), separately. The expression levels of immune response CD markers were measured by flow cytometry (CytoFLEX S, Beckman Coulter, USA).
Tian X., Zeng A., Liu Z., Zheng C., Wei Y., Yang P., Zhang M., Yang F, & Xie F. (2020). Carbon Quantum Dots: In vitro and in vivo Studies on Biocompatibility and Biointeractions for Optical Imaging. International Journal of Nanomedicine, 15, 6519-6529.
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2

Erythropoiesis Characterization Protocol

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Cultured cells were harvested at different time intervals. For morphological characterization, cytospin smears were prepared and stained with Wright’s and Giemsa stain. A total of 500 cells were counted in random non-overlapping fields to determine the different stages of erythropoiesis process.
Phenotypic characterization was done by analyzing the expression of different cell surface markers. Cells were harvested and suspended in 50 μl of 1× phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (Sigma Aldrich, St. Louis MO, USA) and 0.1% azide (Sigma Aldrich, St. Louis MO, USA), and then incubated with specific fluorochrome-tagged antibodies for 45 min at 4 °C in the dark. Proper isotype-matched antibodies were used as controls. The stained cells were washed, re-suspended in PBS, and acquired on FACS Canto II (BD, San Jose, CA, USA). Data were analyzed by using BD FACS DIVA or BD Flow Jo software. The details of the antibodies used are as follows: CD71-FITC, CD235a-APC (ebioscience, San Diego, California, USA), CD235a-Brilliant violet, CD49d-APC, AnnexinV FITC (BD Bioscience, San Jose, CA, USA).
Kuhikar R., Khan N., Philip J., Melinkeri S., Kale V, & Limaye L. (2020). Transforming growth factor β1 accelerates and enhances in vitro red blood cell formation from hematopoietic stem cells by stimulating mitophagy. Stem Cell Research & Therapy, 11, 71.
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3

Imaging Flow Cytometry of Erythroblasts

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Extensively self-renewing erythroblasts or β-yac ESREs were stained with CD71-FITC (eBioscience), c-Kit-PE (eBioscience), DAPI (Sigma-Aldrich, St Louis, MO, USA), and DRAQ5 (eBioscience) and run on the ImageStream (Amnis, Seattle, WA, USA). The data was analyzed with IDEAS software (Amnis) as previously published [24 (link)].
Getman M., England S.J., Malik J., Peterson K., Palis J, & Steiner L.A. (2014). Extensively self-renewing erythroblasts derived from transgenic β-yac mice is a novel model system for studying globin switching and erythroid maturation. Experimental hematology, 42(7), 536-46.e8.
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4

Immunophenotyping of Cultured Cells

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The cells were fixed with 4% paraformaldehyde (PFA) in PBS at room temperature for 10 min, permeabilized with 0.1% Triton X-100 in PBS at room temperature for 10 min, and then incubated with 1% BSA in PBS for 30 min to block nonspecific binding. The treated cells were then incubated with the following antibodies as shown in figures: CD144-Alexa Fluor 488 (eBioscience), CD45-PE (BD Biosciences), CD71-FITC (eBioscience), and CD235a-PE (BioLegend) for one hour at room temperature. The nuclei were stained by 0.1 mg/ml DAPI for 3 min. The results were examined by a fluorescence microscope.
BAI H., LIU Y., XIE Y., HOYLE D.L., BRODSKY R.A., CHENG L., CHENG T, & WANG Z.Z. (2015). Definitive Hematopoietic Multipotent Progenitor Cells Are Transiently Generated From Hemogenic Endothelial Cells in Human Pluripotent Stem Cells. Journal of cellular physiology, 231(5), 1065-1076.
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5

Flow Cytometry Analysis of Hematopoietic Cells

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The cells were harvested, and 2 × 105 cells were placed in aliquots in phosphate-buffered saline (PBS) containing 1% BSA (PBS/BSA) and centrifuged (200g) for 5 minutes. Antibodies were added directly to cell pellets that were then resuspended to a final volume of 100 μl, incubated for 30 minutes on ice, washed in 5 ml PBS/BSA, and resuspended in 300 μl. The samples were analyzed using an LSRFortessa (BD Biosciences, Franklin Lakes, NJ, http://www.bdbiosciences.com) using FACSDiva acquisition (BD Biosciences) and FlowJo analysis software (FlowJo, Ashland, OR, http://www.flowjo.com), or sorted on a FACSARIA (BD Biosciences). Anti-human antibodies included CD71-FITC (catalog no. 11-0719; eBioscience, San Diego, CA, http://www.ebioscience.com), CD43-APC (catalog no. 17-0439-42; eBioscience), glycophorinA (M) (CD235a), efluor450 (catalog no. 48-9884; eBioscience), CD34-PE (catalog no. 12-0349; eBioscience), CD41a-FITC (BD Biosciences), CD45-V450 (BD Biosciences), and CD144-PE (Beckman Coulter, Brea, CA, http://www.beckmancoulter.com). Mouse IgG1 APC isotype control (catalog no. 17-4714-81; eBioscience) and mouse IgG1 PE isotype control (catalog no. 12-4714-81; eBioscience) were used (supplemental online Fig. 2).
Jackson M., Ma R., Taylor A.H., Axton R.A., Easterbrook J., Kydonaki M., Olivier E., Marenah L., Stanley E.G., Elefanty A.G., Mountford J.C, & Forrester L.M. (2016). Enforced Expression of HOXB4 in Human Embryonic Stem Cells Enhances the Production of Hematopoietic Progenitors but Has No Effect on the Maturation of Red Blood Cells. Stem Cells Translational Medicine, 5(8), 981-990.
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6

Splenic Erythrocyte Differentiation Analysis

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Whole spleens were collected, washed in RPMI and PBS and passed through 100 and 70 μM mesh to release cell suspensions. Single cell suspensions obtained from spleens were analyzed with the following antibodies: B220-APC, TER119-PE, CD71-FITC and Annexin V PerCP-eFluor710 (eBioscience). Cells were analyzed on a BD LSRFortessa machine (Becton Dickinson) using FlowJo software. The four different erythrocytic populations were defined according to Socolovsky et al. (2001) (link): I: Ter119medCD71high (pro-erythroblasts); II: Ter119highCD71high (basophilic erythroblasts); III: Ter119highCD71med (late basophilic erythroblasts); and IV: Ter119highCD71low (orthochromatophillic erythroblasts). For compensation, single positive labeled cells and OneComp eBeads (Becton Dickinson) were used. Ki-67 PerCP-eFluor710, the intracellular staining to identify proliferating cells was performed according to the Intracellular Fixation & Permeabilization Buffer Set protocol (eBioscience).
Ilardo M., dos Santos M.C., Grote Beverborg N., Rajan M., Said M.A., Verweij N., Van Der Harst P., Van Der Meer P, & Leibold E.A. (2022). An Erythropoietin-Independent Mechanism of Erythrocytic Precursor Proliferation Underlies Hypoxia Tolerance in Sea Nomads. Frontiers in Physiology, 12, 760851.
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7

Characterization of Macrophage Phenotypes

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PE anti-human CD32 antibody, the mouse IgG2b isotype control, Human M-CSF, CD68-FITC, and the CD86-Alexa-Fluor antibodies were purchased from BioLegend (San Diego, CA). Benzo(a)pyrene [B(a)P] powder, Trypan blue, dimethyl sulfoxide (DMSO), dihydrorhodamine 123, and protease/phosphatase inhibitors were obtained from Sigma-Aldrich (St. Louis, MO). FITC-Dextran (Mr, 40,000 kDa) was purchased from Molecular Probes/Invitrogen (Carlsbad, CA). Phorbol 12-myristate 13-acetate (PMA) and Cholera Toxin Subunit B Hrp-conjugated ab (CTβ) were purchased from Abcam Biochemicals (Cambridge, MA). Specific antibodies to CD11b-PE, CD14-FITC, CD64, CD71-FITC, control IG1Κ and FITC were purchased from eBioscience (San Diego, CA). CD45 and CD59 were purchased from Santa Cruz Biotechnology (Dallas, TX). Amplex Red Cholesterol Assays and Anti-Human IgG(γ)-FITC conjugate ab were obtained from Invitrogen (Carlsbad, CA). Human IgG complexes (human heat-aggregated IgG; hHAIgG) were formed by incubating monomeric human IgG for 20 min at 63°C and centrifuged at 14,000 × g to removed precipitates.
Clark R.S., Pellom S.T., Booker B., Ramesh A., Zhang T., Shanker A., Maguire M., Juarez P.D., Matthews-Juarez P., Langston M.A., Lichtveld M.Y, & Hood D.B. (2016). Validation of Research Trajectory 1 of an Exposome Framework: Exposure to benzo(a)pyrene confers enhanced susceptibility to bacterial infection. Environmental research, 146, 173-184.
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8

Assessing Erythroid Maturation Inhibitors

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Inhibitors utilized for studies of globin expression and erythroid maturation were 1 μmol/L TCP (Sigma), 0.75 μmol/L sodium butyrate (Sigma), 50 μmol/L hydroxyurea (HU) (Sigma) and 0.1 μmol/L decitabine (Selleck Chemicals, Houston, TX, USA). Antibodies used included CD71-FITC (eBioscience, San Diego, CA, USA), and Kit-FITC (eBioscience). Fetal globin detection was done using the Invitrogen Fetal Hemoglobin Test Kit (Invitrogen), according to the manufacturer’s instructions. An APC conjugated fetal hemoglobin antibody (Invitrogen) was substituted for the FITC-conjugated antibody provided in the kit to overcome the autofluorescence inherent in cultured cells.
Getman M., England S.J., Malik J., Peterson K., Palis J, & Steiner L.A. (2014). Extensively self-renewing erythroblasts derived from transgenic β-yac mice is a novel model system for studying globin switching and erythroid maturation. Experimental hematology, 42(7), 536-46.e8.
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9

Baclofen Treatment on Murine Hematopoietic Stem Cells

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C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) were maintained in an animal facility. Animal care followed MDACC Institutional Animal Care and Use Committee oversight and guidelines. Mice around 10 weeks old were treated with baclofen (Millipore Sigma, St. Louis, MO) at the dose of 3 mg/kg body weight or with phosphate-buffered saline vehicle control (0.2 mL, i.p.) daily for 1 week. Mice were then euthanized with CO 2 , and BM cells were isolated. BM cells were analyzed by flow cytometry using a Gallios flow cytometer (Beckman Coulter, Indianapolis, IN). The Lin -/Sca1 + /cKit1 + (LSK) BM cells were isolated by fluorescence-activated cell sorting (FACS). Isolated LSK cells were plated into mouse Methocult-3344 (Stem Cell Technology) to culture for 10-14 days before the counting of colonies. The antibodies used for mouse BM cell analysis and LSK isolation included biotin mouse lineage cocktail (Miltenyi 130-090-858), streptavidin-APC-C7 (554063, BD Pharmingen, East Rutherford, NJ), cKit-APC (553356, BD Pharmingen), Sca1-PerCP (45-5981-82, eBioscience, San Diego, CA), CD71-FITC (11-0314-85, eBioscience), and Ter119-eFluor450 (48-5921-82, eBioscience).
Darbaniyan F., Zheng H., Kanagal-Shamanna R., Lockyer P., Montalban-Bravo G., Estecio M., Lu Y., Soltysiak K.A., Chien K.S., Yang H., Sasaki K., Class C., Ganan-Gomez I., Do K.A., Garcia-Manero G, & Wei Y. (2022). Transcriptomic Signatures of Hypomethylating Agent Failure in Myelodysplastic Syndromes and Chronic Myelomonocytic Leukemia. Experimental hematology, 115.
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