Anti mouse cd71 pe

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Anti-mouse CD71-PE is a fluorescently labeled antibody that binds to the CD71 (transferrin receptor) antigen expressed on the surface of mouse cells. It is used for the identification and enumeration of CD71-positive cells in flow cytometry applications.

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7 protocols using anti mouse cd71 pe

Multiparameter Flow Cytometry Analysis

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All flow cytometric data were acquired on a Fortessa flow cytometer (BD Biosciences) and analyzed using Flowjo software (Tree Star). All stainings were carried out in FACS buffer (2mM EDTA and 1% FBS in phosphate-buffered saline/PBS) for 20 minutes on ice. Samples were washed twice with FACS buffer prior to flow analyses. The following are the antibodies used, all at 1:100 dilution: anti-myc tag-PE (Cell Signaling Technology, 3739), anti-mouse Ter119-APC (eBioscience, 17-5921-83), and anti- mouse CD71-PE (Affymetrix, 12-0711-83). Hoechst 33342 (Life Technologies, H1399) was used to visualize nuclei.

Deshycka R., Sudaryo V., Huang N.J., Xie Y., Smeding L.Y., Choi M.K., Ploegh H.L., Lodish H.F, & Pishesha N. (2021). Engineered red blood cells carrying PCSK9 inhibitors persistently lower LDL and prevent obesity. PLoS ONE, 16(11), e0259353.

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Multiparameter Flow Cytometry of Erythroid Cells

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All flow cytometric data were acquired on a fluorescence-activated cell sorter (FACS) Fortessa flow cytometer (BD Biosciences) and analyzed using the Flowjo software (Tree Star). All stainings were carried out in FACS buffer (2 mM EDTA and 5% FBS in phosphate-buffered saline (PBS)) for 40 min at room temperature unless otherwise described. Samples were washed twice with FACS buffer prior to flow analyses. The following are the antibodies used at 1:100 dilution: anti-human CD235A-APC (eBioscience, 17-9987042), anti-human CD71-FITC (eBioscience, 11-0719-42), anti-human CD71-PeCy7 (Affymetrix, 25-0719-42), anti-human CD117-PeCy7 (eBioscience, 25-1178-42), anti-human CD117-BV605 (BioLegend, 313217), anti-human CD49e-APC (BioLegend, 328012), anti-human CD29-PerCP-eFluor 710 (Affymetrix, 46-0299-41), anti-human CD49d-PE (Affymetrix, 12-0499-42), anti-human CD240DCE-APC (Miltenyi Biotec, 130-104-818), anti-human CD238-APC (Miltenyi Biotec, 130-104-951), anti-human CD47-PerCP-eFluor 710 (Affymetrix, 46-0479-42), anti-human CD147-PE(Affymetrix, 12-1472-42), anti-human CD59-APC (Affymetrix, 17-0596-42), anti-myc tag-PE (Cell Signaling Technology, 3739), anti-mouse Ter119-APC (eBioscience, 17-5921-83), and anti-mouse CD71-PE (Affymetrix, 12-0711-83). Hoechst 33342 (Life Technologies, H1399) was used to visualize nuclei.

Huang N.J., Pishesha N., Mukherjee J., Zhang S., Deshycka R., Sudaryo V., Dong M., Shoemaker C.B, & Lodish H.F. (2017). Genetically engineered red cells expressing single domain camelid antibodies confer long-term protection against botulinum neurotoxin. Nature Communications, 8, 423.

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Murine Erythroid Cell Isolation and Analysis

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Biotin-X-NHS (N-hydroxysuccinimide ester of biotin) was obtained from Calbiochem (La Jolla, CA). Streptavidin Allophycocyanin (SAv-APC), rat anti-mouse Ter-119-APC, rat anti-mouse Ter-119-FITC, anti-mouse CD16/CD32 were from BD Biosciences (San Diego, CA, USA). Anti-mouse CD71-PE, anti-mouse CD71-biotin, Annexin-V-FITC, anti-mouse IgG1κ-PE, anti-mouse IgG2bκ-APC, anti-mouse IgG2bκ-FITC, 7-Aminoactinomycin D (7-AAD) were procured from e-biosciences (San Diego, CA, USA). 5 (and 6) chloromethyl-2, 7 dichloro-dihydro-fluorescein diacetate (CM-H₂DCFDA) was purchased from Molecular Probes (Eugene, OR, USA). Mouse Easy Sap biotin selection kit was procured from Stem cell technology (USA). Revert Aid First strand cDNA synthesis kit was from Thermo Scientific (India). Power SYBR green PCR Master mix was from Applied Biosystem (CA, USA). Fetal bovine serum was obtained from Hyclone (South Logan, UT). TRI reagent, RPMI, HEPES, Thiazole Orange (TO), Dimethylformamide (DMF), Paraquat and other analytical reagents were from Sigma-Aldrich (India).

Bhardwaj N, & Saxena R.K. (2014). Elimination of Young Erythrocytes from Blood Circulation and Altered Erythropoietic Patterns during Paraquat Induced Anemic Phase in Mice. PLoS ONE, 9(6), e99364.

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Immunophenotyping of Human and Mouse Erythroid Cells

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Cells were harvested and washed three times with PBS. Then, 5 μL per 1 × 10⁶ cells were stained with either anti-human CD71-APC (BD Biosciences, Franklin, NJ, USA) or anti-human CD71-FITC (BD Biosciences), anti-human CD235a-PE (BD Biosciences), anti-human α4 integrin (CD49d-PE, eBioscience, San Diego, CA, USA), anti-human Band 3-APC (graciously provided by Professor Xiuli An) or anti-mouse CD71-PE (eBioscience) and anti-mouse Ter119-APC at 4 °C for 40 min. Subsequently, the cells were washed with PBS or normal saline (NS), followed by analysis using a FACS Calibur machine (BD Biosciences).

Fang F., Xu L., Liang L., Qu M., Yao H., Yue W., Chen L., Chen D., Fan Z., He L., Nan X., Zhang H., Xie X, & Pei X. (2022). The accumulation of miR-125b-5p is indispensable for efficient erythroblast enucleation. Cell Death & Disease, 13(10), 886.

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Comprehensive Flow Cytometry Staining Protocol

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All flow cytometry data were acquired using either using LSR II SORP or LSR Fortessa flow cytometers (BD Biosciences). All staining was carried out in FACS buffer (2% FBS in PBS) for 30 min on ice unless otherwise described. The following antibodies were used anti-human CD235a-APC (eBioscience, Clone HIR2), anti-human CD71-FITC (eBioscience, Clone OKT9), anti-human CD71-PEcy7 (eBioscience, Clone OKT9), ant-human CD49d-PE (Miltenyi, Clone MZ18-24A9), anti-human CD41a-PE (eBioscience, Clone HIP8), anti-human CD11b-PE (eBioscience, Clone ICRF44), anti-mouse Ter119-APC (eBioscience, Clone TER119), anti-mouse CD71-PE (eBioscience, Clone R17217) and Alexa Fluor-647 anti-phospho STAT5 (pY694) (BD Bioscience Cat#: 612599). Hoechst 33342 (Life Technologies, H1399) was used to visualize nuclei.

Nandakumar S.K., McFarland S.K., Mateyka L.M., Lareau C.A., Ulirsch J.C., Ludwig L.S., Agarwal G., Engreitz J.M., Przychodzen B., McConkey M., Cowley G.S., Doench J.G., Maciejewski J.P., Ebert B.L., Root D.E, & Sankaran V.G. (2019). Gene-centric functional dissection of human genetic variation uncovers regulators of hematopoiesis. eLife, 8, e44080.

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Platelet Characterization Antibody Panel

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The following antibodies from eBioscience were used for FCM: FITC-anti-mouse-CD41, APC-anti-mouse-CD42, PE-anti-mouse-CD71, PE-anti-human-CD34, APC-anti-human-CD61, FITC-anti-mouse-CD34 and PE/FITC- anti-human-CD41a. A rat anti-mouse-CD41 monoclonal antibody (Abcam) diluted 1:50 in PBS was used for immunohistochemical staining. CD63 mouse monoclonal antibody (Santa Cruz Biotechnology), RHOB monoclonal antibody (ABclonal) and GAPDH monoclonal antibody (Cell Signaling Technology) were diluted 1:1000 and used for Western blotting, and 1 μg per mL human TPO antibody or human TPO receptor antibody (R&D Systems) was used to neutralize TPO in PMPs. The information on the antibodies used in our study is provided in Supplementary Table 2.

Qu M., Zou X., Fang F., Wang S., Xu L., Zeng Q., Fan Z., Chen L., Yue W., Xie X, & Pei X. (2020). Platelet-derived microparticles enhance megakaryocyte differentiation and platelet generation via miR-1915-3p. Nature Communications, 11, 4964.

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Erythroid Precursor Quantification in Mouse Bone Marrow

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Bone marrow cells flushed out from the hind limbs of mice were filtered through a mesh. The single-cell suspensions of 2 × 10⁶ cells in PBS with 2% fetal calf serum were blocked with CD16/32 antiobdy (1 μg/ml; eBioscience, San Diego, CA, USA) on ice for 10 min, and then stained with PE-anti-mouse CD71 (1 μg/ml; eBioscience) and FITC-anti-mouse Ter119 (1 μg/ml; eBioscience) for 30 min. Dead cells and nonspecific signals were excluded by propidium iodide (Sigma, St. Louis, MO, USA) staining and appropriate isotype controls (eBioscience). A BD FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) was used to detect the percentage of erythroid precursor cells at different maturation stages. For the colony formation units counting, the another part of the BM cells were incubated in ACK lysis buffer for 10 min at room temperature to obtain the nucleated cells. The 2 × 10⁴ BM nucleated cells were embedded in 1 ml of Stemcell M3434 or M3334 semi-solid medium (StemCell Technologies, Vancouver, Canada) at 37°C, 5% CO₂ for 10 or 2 days, and then colonies numbers of burst-forming units-erythroid (BFU-E) and colony formation units-erythroid (CFU-E) were observed, respectively.

Liu Y.S., Huang H., Zhou S.M., Tian H.J, & Li P. (2018). Excessive Iron Availability Caused by Disorders of Interleukin-10 and Interleukin-22 Contributes to High Altitude Polycythemia. Frontiers in Physiology, 9, 548.

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