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Anti cd235a

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Anti-CD235a is a laboratory reagent used to detect the presence of the CD235a antigen, also known as glycophorin A, on the surface of red blood cells. It is commonly used in flow cytometry and other analytical techniques to identify and characterize red blood cell populations.

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Lab products found in correlation

Anti cd14, by BD (3 mentions) Anti cd45, by BD (3 mentions) Anti cd16, by Miltenyi Biotec (2 mentions) Anti cd61, by BD (2 mentions) Anti cd41, by BD (2 mentions) Anti cd43, by BD (2 mentions) Anti cd34, by BD (2 mentions) Accuri c6, by BD (2 mentions) Cellquest pro software 5, by BD (1 mentions) Apc labelled annexin 5, by BD (1 mentions) Trucount tube, by BD (1 mentions) Facscanto 2, by BD (1 mentions) Anti cd8, by BD (1 mentions) Anti cd56, by BD (1 mentions) Anti cd15, by BD (1 mentions) Anti tcrαβ, by BD (1 mentions) Anti cd7, by BD (1 mentions) Anti cd5, by BD (1 mentions) Facscanto, by BD (1 mentions) Anti cd3, by BD (1 mentions)

9 protocols using anti cd235a

1

Flow Cytometric Evaluation of Leukemic Cell Differentiation

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Indirect immunofluorescence was used to detect the expression of differentiation- associated antigens on the surface of leukemic cells. Cells on days 1, 3, and 5 were washed with phosphate buffered saline (PBS) and incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (goat F(ab’)2 anti-mouse IgG, (Cappel Laboratories, Cochranville, PA, USA) with primary monoclonal antibodies. Differentiated antigens were used to detect differentiation. Examples included anti-CD14 (BD Biosciences) for monocytes [50 (link)], anti-CD16 [51 (link)] (Miltenyi Biotec, Bergisch Gladbach, Germany) for neutrophils, anti-CD235a (BD Biosciences) for erythrocytes, and anti-CD61 [52 (link)] (BD Biosciences) for megakaryocytes. The background threshold was obtained using a FITC-conjugated goat anti-mouse IgG antibody. Flow cytometry was conducted using a FACSCalibur with an Argon-ion laser and a 488 nm emission filter, and the data were analyzed using the CellQuestPro software 5.1 (Becton Dickinson).
Lin J.F., Chi C.W., Huang Y.C., Tsai T.H, & Chen Y.J. (2023). Anti-Cancer Effects of Oxygen-Atom-Modified Derivatives of Wasabi Components on Human Leukemia Cells. International Journal of Molecular Sciences, 24(7), 6823.
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2

Flow Cytometric Analysis of Microparticles

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MPs were labelled for flow cytometric analysis as previously described.30 (link) Briefly, for the determination of the cellular source of MPs, 25 µL of freshly thawed FFP were mixed with 2.5 µL of fluorescein isothiocyanate (FITC)-labelled anti-CD41 (platelet marker) or phycoerythrin (PE)-labelled anti-CD235a (RBC marker) (BD Biosciences, San Jose, CA). Phosphatidylserine (PS) expression on MPs was determined by labelling 12.5 µL of FFP with 5 µL of allophycocyanin (APC) labelled-annexin-V (BD Biosciences) or FITC labelled-lactadherin (Haematologic Technologies, Essex Junction, Vermont). Samples were diluted with 0.2 µm-filtered phosphate buffered saline (PBS), pH 7.2 or annexin-binding buffer to a final reaction volume of 100 µL in an absolute count tube (TruCount tubes, BD BioSciences). After incubation in the dark at RT for 30 minutes, 300 µL of PBS or annexin-binding buffer was added to each sample. Concentration-matched isotype antibodies were used as controls. Analyses were performed on a FACSCantoII (BD Biosciences) with instrument settings and gating optimized for detection of MPs less than 1 µm diameter as previously described.30 (link) Absolute count of MPs was calculated according to the manufacturer’s instructions (BD Biosciences).
Chan K.S, & Sparrow R.L. (2014). Microparticle Profile and Procoagulant Activity of Fresh Frozen Plasma is Affected by Whole Blood-Leukocyte Depletion Rather Than 24-Hour Room Temperature-Hold. Transfusion, 54(8), 1935-1944.
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3

Flow Cytometric Phenotyping of Immune Cells

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The following conjugated antibodies were used for flow cytometric phenotyping and analysis: anti-CD34, anti-CD43, anti-KDR, anti-CD45, anti-CD7, anti-CD5, anti-CD4, anti-CD8, anti-CD3, antiTCRαβ, anti-CD56, anti-CD15, anti-CD14, and anti-CD235a purchased from BD Biosciences. All antibodies were used in a 1:30 dilution. Dead cells were excluded from analysis in all experiments by staining with DAPI. Flow cytometry analysis was conducted on an LSRII cytometer (BD Biosciences, Paris, France), a FACS CANTO (BD Biosciences, Paris, France), or a FACS CELESTA (BD Biosciences, Paris, France) and analyzed using FlowJo software (BD Biosciences, Ashland, OR, USA).
Flippe L., Gaignerie A., Sérazin C., Baron O., Saulquin X., Anegon I., David L, & Guillonneau C. (2022). Generation of CD34+CD43+ Hematopoietic Progenitors to Induce Thymocytes from Human Pluripotent Stem Cells. Cells, 11(24), 4046.
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4

Comprehensive Red Blood Cell Analysis

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At each time point, RBC concentration, Hct level, HbTot, MCV, and MCHC were assessed using a hematology analyzer (Coulter AcT 5diff AL, Beckman Coulter). The hemolysis was calculated with the following equation: Hemolysis = ([fHb]/[HbTot]) * (100 – Hct). The [ATP] was measured for RCCs using the ATPlite kit (Perkin Elmer). The [glucose], [lactate], [K+], and [Na+] were measured using a blood analyzer (ABL90 FLEX PLUS). Serial dilutions in saline were carried out if the results were greater than the detection limits of the apparatus. Alexafluor 488 AnnexineV (A13201, Invitrogen) and Anti-CD235a (551336, BD Biosciences) were used to detect PS exposure by flow cytometry (Accuri C6, BD Biosciences).
Robidoux J., Laforce-Lavoie A., Charette S.J., Shevkoplyas S.S., Yoshida T., Lewin A, & Brouard D. (2020). Development of a flow standard to enable highly reproducible measurements of deformability of stored red blood cells in a microfluidic device. Transfusion, 60(5), 1032-1041.
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5

Erythroid Progenitor Cell Analysis

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On day 8, 10, 12, 14 and 16 time points, non-adherent cells were collected and washed in IMDM. Cells were stained with cell surface antibodies, anti CD235a cat# 551336 (BDbiosciences, San Jose, CA, USA) and anti CD71 cat# 555537 (BDbiosciences, San Jose, CA, USA) for 30 min at 4 °C. Next cells were fixed and permeabilized with 200 ul of Cytospin/Cytoperm (BD Biosciences San Jose, CA) followed by intracellular staining using anti AHSP cat# sc515436 (Santa Cruz Biotechnology, Dallas, TX, USA) for 2 h at room temperature, and then with Hoechst 33342 nuclear stain cat# H3570 (Thermofisher Invitrogen, Waltham, MA) for 10 min at room temperature. Cells were then analyzed by flow cytometry using BD LSRFortessa and gated with FlowJo software.
Walczak J., Camargo Johnson M.D, & Muthumalaiappan K. (2020). Stage Specific Expression Pattern of Alpha-Hemoglobin-Stabilizing-Protein (AHSP) Portrayed in Erythroblast Chronology. Methods and Protocols, 3(3), 46.
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6

Megakaryocyte Differentiation Analysis

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Meg01, Meg01-MEF2CKO, CHOP10, CHOP10-MEF2C-KO, and CB-derived megakaryocytes were treated as described above. Cells were stained with anti-CD34 (Biolegend, San Diego, CA), anti-CD235a, anti-CD45, anti-CD41, or anti-CD42 antibodies (BD Biosciences, San Jose, CA) and then analyzed by flow cytometry using an Accuri C6 flow cytometer (BD Biosciences).
Kong X., Ma L., Chen E., Shaw C.A, & Edelstein L.C. (2019). Identification of the Regulatory Elements and Target Genes of Megakaryopoietic Transcription Factor MEF2C. Thrombosis and haemostasis, 119(5), 716-725.
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7

Immune Profiling of Hematopoietic Differentiation

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During hematopoietic differentiation and erythroid maturation, cells were collected for immunophenotyping. At least 105 cells were incubated with antibodies (1:20) for 15 min and then washed with 2 mL of phosphate-buffered saline (PBS) 1×. The cell suspension was centrifuged for 3 min at 330 × g. The pellet obtained was resuspended at 200 µL and then acquired (10,000 events) on flow cytometry FACSCalibur or FACSAria (Becton Dickinson). The following antibodies (BD Bioscience) were used: anti-CD34, anti-CD36, anti-CD43, anti-CD45 anti-CD71, anti-CD235a, anti-NANOG, anti-SOX-2 and anti-OCT3/4. Data obtained were analyzed using the softwares FACSDiva and CELLQuest™ (Becton Dickinson).
Paes B.C., Stabeli L.C., Costa P.N., Orellana M.D., Kashima S., Covas D.T, & Picanço-Castro V. (2020). Generation of hematopoietic stem/progenitor cells with sickle cell mutation from induced pluripotent stem cell in serum-free system. Hematology, Transfusion and Cell Therapy, 43(2), 156-164.
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8

Phenotypic Characterization of Cell Populations

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The cells diluted in HBSS were stained with the following human antibodies for cell sorting and cell surface analysis: phycoerythrin (PE)-conjugated anti-CD73 (BioLegend, San Diego, CA), allophycocyanin (APC)-conjugated anti-CD29, anti-CD44, APC-conjugated anti-CD73, fluorescein isothiocyanate (FITC)-conjugated anti-CD90, PE-conjugated anti-CD271, PE-Cy7-conjugated anti-CD31, anti-CD45, and anti-CD235a (BD, Franklin Lakes, NJ). Propidium iodide fluorescence was used to gate dead cells. Flow cytometry and sorting were performed on a FACS Aria II instrument (BD).
Suto E.G., Mabuchi Y., Toyota S., Taguchi M., Naraoka Y., Itakura N., Matsuoka Y., Fujii Y., Miyasaka N, & Akazawa C. (2020). Advantage of fat-derived CD73 positive cells from multiple human tissues, prospective isolated mesenchymal stromal cells. Scientific Reports, 10, 15073.
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9

Leukemic Cell Surface Antigen Detection

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An indirect immunofluorescence method was used to detect the expression of differentiation-associated antigens on the surface of leukemic cells. Cells collected from day-5 cultures were cultured with primary monoclonal antibodies, washed with PBS, and exposed to FITC-conjugated secondary antibodies, i.e., goat F(ab’)2 anti-mouse IgG (Cappel Laboratories, Cochranville, PA, USA). Monoclonal antibodies used included anti-CD14 (BD Biosciences) for monocyte, anti-CD16 (Miltenyi Biotec, Bergisch Gladbach, Germany) for neutrophil, anti-CD235a (BD Biosciences) for erythrocyte, and anti-CD61 (BD Biosciences) for megakaryocyte. FITC conjugated to goat anti-mouse IgG was used to set background thresholds. Flow cytometry with FACScaliber and data analysis using CellQuest Pro software (Becton Dickinson) were carried out.
Wu K.M., Liao H.F., Chi C.W., Kou Y.R, & Chen Y.J. (2019). Wasabi Compound 6-(Methylsulfinyl) Hexyl Isothiocyanate Induces Cell Death with Coexisting Mitotic Arrest and Autophagy in Human Chronic Myelogenous Leukemia K562 Cells. Biomolecules, 9(12), 774.
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