Cd235a apc cy7

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CD235a-APC-Cy7 is a fluorochrome-conjugated antibody that binds to the CD235a (Glycophorin A) antigen. CD235a is a sialoglycoprotein expressed on the surface of erythroid cells. This antibody can be used for the identification and enumeration of erythroid cells in flow cytometry applications.

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APC-Cy7 (106 citations)

4 protocols using cd235a apc cy7

Single-cell RNA-seq of Tumor Samples

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Fresh isolated tumour samples were dissociated using the human tumour dissociation kit (Miltenyi Biotec; 130–095-929), sorted into 96-well plates containing 10 μl of TCL buffer (Qiagen) with 1% β-mercaptoethanol, using the following anti-human antibodies: FcX (Biolegend, 422302), CD45-PE (Biolegend, 304008), CD3-APC (Biolegend, 300412), CD235a-APC/Cy7 (Biolegend, 349116) and HLAA,B,C-FITC (Biolegend, 311426). Sorting of viable cells was performed using the live/dead dye Zombie Violet (Biolegend, 77477). Single-cell libraries were generated using a modified version of the full-length Smart-seq2 protocol as previously described⁶¹, and were sequenced on a NextSeq 500 sequencer (Illumina), resulting in a median of approximately 1.4 million paired-end reads and a median of 2,588 genes detected per cell. A cutoff of log₂(transcripts per million (TPM) + 1) ≥ 2 was used to define a gene as expressed in each single cell. For each sample, we computed the fraction of B cells using pre-defined markers (CD19 and/or MS4A1). Notably, this is a plate-based protocol; thus, for each patient, we collected and sequenced the same number of cells (n = 384 CD45⁺ cells per plate). Thus, the number of cells per patient is equal, and the frequency reflects patients with either high or low B cell infiltrate.

Helmink B.A., Reddy S.M., Gao J., Zhang S., Basar R., Thakur R., Yizhak K., Sade-Feldman M., Blando J., Han G., Gopalakrishnan V., Xi Y., Zhao H., Amaria R.N., Tawbi H.A., Cogdill A.P., Liu W., LeBleu V.S., Kugeratski F.G., Patel S., Davies M.A., Hwu P., Lee J.E., Gershenwald J.E., Lucci A., Arora R., Woodman S., Keung E.Z., Gaudreau P.O., Reuben A., Spencer C.N., Burton E.M., Haydu L.E., Lazar A.J., Zapassodi R., Hudgens C.W., Ledesma D.A., Ong S., Bailey M., Warren S., Rao D., Krijgsman O., Rozeman E.A., Peeper D., Blank C.U., Schumacher T.N., Butterfield L.H., Zelazowska M.A., McBride K.M., Kalluri R., Allison J., Petitprez F., Fridman W.H., Sautès-Fridman C., Hacohen N., Rezvani K., Sharma P., Tetzlaff M.T., Wang L, & Wargo J.A. (2020). B cells and tertiary lymphoid structures promote immunotherapy response. Nature, 577(7791), 549-555.

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Multicolor Flow Cytometry of THP-1 and Blood

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Flow cytometry analysis of THP-1 cells and total blood was performed using the following antibodies. Conjugated anti-human SR-B1 APC (Miltenyi Biotec 130-111-237), CD235a APC Cy7 (BioLegend 3,49,115), CD45 BUV805 (BD Biosciences 6,12,892), CD56 BUV 737 (BD Biosciences 6,12,767), CD11c PeCy7 (BioLegend 3,37,216), CD11b PE (eBioscience 12-0118-41), CD3 PerCP Cy5.5 (BD Biosciences 5,60,835), CD19 BV786 (BioLegend 3,02,239), HLA-DR BUV 395 (BD Biosciences 7,40,302), CD14 BV605 (BioLegend 3,01,834). Labeling of mouse blood was done using the following conjugated antibodies: CD45.2 PE (eBioscience 12-0454–82), CD115 PerCPeF710 (eBioscience 46-1,152-82), CD11b BV650 (eBioscience 48-0112–20), Ly6G BV510 (BD Biosciences 5,63,402), CD3 APC-eF780 (BioLegend 47-0032–82). Fc receptors were blocked using FCR blocking reagent (Myltenyi Biotec). Surface membrane staining was performed in PBS FBS 2%. The Zombie UV fixable viability dye (BioLegend 4,23,107) was used to exclude dead cells. Samples were acquired on a Cytoflex (Beckman Coulter) and analyzed with FlowJo 10 (BD Biosciences).

Tello Rubio B., Bugault F., Baudon B., Raynal B., Brûlé S., Morel J.D., Saint-Auret S., Blanchard N., Demangel C, & Guenin-Macé L. (2021). Molecular Mechanisms Underpinning the Circulation and Cellular Uptake of Mycobacterium ulcerans Toxin Mycolactone. Frontiers in Pharmacology, 12, 733496.

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Characterizing Blood Cell Populations

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Plasma particles were incubated with 20 μL of Human TruStain FcX Fc Receptor Blocking Solution (Biolegend) for 10 min at room temperature. Fluorescently labeled antibodies (CD41-PE, CD45-PerCP, and CD235a-APC-Cy7 from BioLegend) were subsequently added to the samples and incubated at room temperature for 30 min in the dark. Samples were washed twice with Tyrode’s buffer by centrifugation at 800 rcf for 5 min at room temperature. Flow cytometry analysis was conducted on a Thermo Fisher Attune NxT at the Cornell BRC Flow Cytometry Facility.

Vu L.T., Ahmed F., Zhu H., Iu D.S., Fogarty E.A., Kwak Y., Chen W., Franconi C.J., Munn P.R., Tate A.E., Levine S.M., Stevens J., Mao X., Shungu D.C., Moore G.E., Keller B.A., Hanson M.R., Grenier J.K, & Grimson A. (2024). Single-cell transcriptomics of the immune system in ME/CFS at baseline and following symptom provocation. Cell Reports Medicine, 5(1), 101373.

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Multiparametric Flow Cytometry of Vascular Cells

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The following antibodies were used: CD45-APC-Cy7 (BD, 557833, 1:50), CD31-Biotin (eBioscience, 13-0319-82, 1:50), Steptavidin-APC-eFlour780 (eBioscience, 47-4317-82, 1:100), CD235a-APC-Cy7 (Biolegend, 349116, 1:50), CD140a-BB515 (BD, 564594, 1:50), PDPN-APC (eBioscience, 17-9381-41, 1:50) and CADM1-PE (MBL, CM004-5, 1:50). Cells were stained in sorting buffer (PBS + 1% BSA) for 30 min at 4 °C, washed once and resuspended in sorting buffer with 7-AAD (eBioscience, 00-6993-50, 1:50) as live cell dye. Flow cytometry was performed on BD FACS Aria II. Pre-gating was first done for live cells based on 7-AAD staining. Gating strategies were based on Fluorescence Minus One (FMO) controls. FlowJo v10 software was used for analyzing the flow cytometry data.

He J., Yan J., Wang J., Zhao L., Xin Q., Zeng Y., Sun Y., Zhang H., Bai Z., Li Z., Ni Y., Gong Y., Li Y., He H., Bian Z., Lan Y., Ma C., Bian L., Zhu H., Liu B, & Yue R. (2021). Dissecting human embryonic skeletal stem cell ontogeny by single-cell transcriptomic and functional analyses. Cell Research, 31(7), 742-757.

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