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Cd45ra bv650

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CD45RA BV650 is a fluorescent-conjugated antibody used in flow cytometry to identify and quantify CD45RA-positive cells. It binds to the CD45RA isoform of the CD45 cell surface glycoprotein.

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Lab products found in correlation

Ccr7 bv421, by BioLegend (2 mentions) Hla dr pe, by BD (2 mentions) Cd8 pe cy7, by BD (2 mentions) Cd4 af700, by BD (2 mentions) Cd19 bv605, by BD (2 mentions) Cd16 apc fire750, by BD (2 mentions) Cd56 af647, by BD (2 mentions) Ccr7 pe dazzle 594, by BD (2 mentions) Cd45 a488, by BD (2 mentions) Cd3 bv785, by BD (2 mentions) Zombie aqua fixable viability kit, by BD (2 mentions) Cd14 percp cy5, by BD (2 mentions) Cd3 v500, by BD (1 mentions) Cd127 bv421, by BioLegend (1 mentions) Lsrfortessa, by BD (1 mentions) Foxp3 transcription factor staining kit, by Thermo Fisher Scientific (1 mentions) Cd28 pe cy7, by BD (1 mentions) Cd27 apc efluor780, by Thermo Fisher Scientific (1 mentions) Cd4 buv737, by BD (1 mentions) Cd27 bv510, by BioLegend (1 mentions)

7 protocols using cd45ra bv650

1

Multiparametric Immune Cell Profiling

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PBMCs were labeled with different combinations of the following antibodies: CD4 BUV737 (BD, clone SK3), CD8 BV786 (BD, clone RPA-T8), CD8 BUV805 (BD, clone SK1), CD3 V500 (BD, clone UCHT1), CD3 eVolve605 (eBioscience clone OKT3), CD27 APC-eFluor780 (eBioscience, clone), CD27 BV510 (Biolegend, clone O323), CD45RA BV650 (BD, clone HI100), CD45RA Qdot655 (Invitrogen, clone MEM-56), CD28 PE-Cy7 (BD, clone 28.2), CX3CR1 APC (eBioscience, clone 2A9-1), CCR7 BV421 (Biolegend, clone G043H7), CD127 BV421 (Biolegend, clone A019D5). Near-IR fixable dye (Invitrogen) was used to exclude dead cells from the analysis. For intracellular staining, the following antibodies were used: Hobit IgM (BD, clone Sanquin-Hobit/1), Eomes eFluor660 (eBioscience, clone WD1928), Tbet BV421 (Biolegend, clone 4B10), Granzyme B AF700 (BD, clone GB11), Perforin FITC (eBioscience, clone dG9), Perforin PE (eBioscience, clone B-D48), Granzyme K PE (Immunotools, clone 24C3), Granzyme K FITC (Immunotools, clone 24C3). To stain for Hobit IgM, a secondary anti-IgM labeled with PE or FITC was used. The cells were labeled according to manufacturer’s instructions. For the intracellular staining, the cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining kit (eBioscience). The samples were measured in PBS 0.5% FCS with a LSR Fortessa (BD). The analysis was done using FlowJo Version 10 software.
Oja A.E., Vieira Braga F.A., Remmerswaal E.B., Kragten N.A., Hertoghs K.M., Zuo J., Moss P.A., van Lier R.A., van Gisbergen K.P, & Hombrink P. (2017). The Transcription Factor Hobit Identifies Human Cytotoxic CD4+ T Cells. Frontiers in Immunology, 8, 325.
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2

Multiparametric Immune Cell Profiling

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All antibodies are from BioLegend (San Diego) unless otherwise noted: CD16-APC-fire750, clone: 3G8; CD56-AF647, clone: NCAM; CD45RA-BV650, clone: HI100; CCR7-Pe/Dazzle 594, clone: G043H7; CD8-PeCy7, clone: SK1; CD45-A488, clone: SD1; CD4-AF700, clone: OKT4; CD19-BV605, clone: HIB19; CD3-BV785, clone: OKT3; HLADR-PE, clone: L243 (BD Biosciences, CA); CD14-PerCP-Cy5.5, clone: MφP9 (BD Biosciences, CA); Zombie Aqua™ Fixable Viability Kit.
Unterberg M., Ehrentraut S.F., Bracht T., Wolf A., Haberl H., von Busch A., Rump K., Ziehe D., Bazzi M., Thon P., Sitek B., Marcus K., Bayer M., Schork K., Eisenacher M., Ellger B., Oswald D., Wappler F., Defosse J., Henzler D., Köhler T., Zarbock A., Putensen C.P., Schewe J.C., Frey U.H., Anft M., Babel N., Steinmann E., Brüggemann Y., Trilling M., Schlüter A., Nowak H., Adamzik M., Rahmel T, & Koos B. (2023). Human cytomegalovirus seropositivity is associated with reduced patient survival during sepsis. Critical Care, 27, 417.
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3

T Cell Phenotyping for Memory Subsets

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The following T cell phenotyping panel was used to determine Tcm, Tem, Tscm and Tnaive populations - CD3 PE-Cy7 (Clone UCHT1), CD4 APC-Cy7 (Clone RPA-T4), CD8 APC (Clone RPA-T8), CD62L BV510 (Clone DREG-56), CD45RA BV650 (Clone HI100), CD95 BV711 (Clone DX2) (BD Biosciences).
Houghton B.C., Panchal N., Haas S.A., Chmielewski K.O., Hildenbeutel M., Whittaker T., Mussolino C., Cathomen T., Thrasher A.J, & Booth C. (2022). Genome Editing With TALEN, CRISPR-Cas9 and CRISPR-Cas12a in Combination With AAV6 Homology Donor Restores T Cell Function for XLP. Frontiers in Genome Editing, 4, 828489.
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4

Quantifying SARS-CoV-2 Spike-specific T cells

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Frequencies of S-specific memory T cells in blood and BAL were assessed using a re-stimulation assay as described previously using 2 μg/mL PepMix SARS-CoV-2 Spike overlapping peptides in DMSO (15mers with 11 amino acid overlap, JPT Peptide Technologies)51 (link). The antibody panel used for surface staining was: CD103 FITC (2G5, Beckman Coulter, cat# B49222, 1:50 dilution), CCR7 BV421 (G043H7, Biolegend, cat# 353208, 1:50 dilution), CD8a BV711 (RPA-T8, Biolegend, cat# 301044, 1:80 dilution), CD4 PE-Cy5.5 (S3.5, Invitrogen, cat# MHCD0418, 1:80 dilution) and CD45RA BV650 (5H9, BD, cat# 740608, 1:500 dilution), and the intracellular proteins were stained using: IL-21 AF647 (3A3-N2.1, BD, cat# 560493, 1:20 dilution), IL-13 PE (JES10-5A2, BD, cat# 559328, 1:33 dilution), IL-2 BV605 (MQ1-17H12, BD, cat# 564165, 1:50 dilution), IL-17A BV785 (BL168, Biolegend, cat# 512338, 1:67 dilution), CD69 ECD (TP.1.55.3, Beckman Coulter, cat# 6607110, 1:67 dilution), CD3 APC-Cy7 (SP34-2, BD, cat# 557757, 1:200 dilution) and IFNγ AF700 (B27, Biolegend, cat# 506516, 1:200 dilution). Acquisition was performed using BD LSRFortessa cell analyzer, and the data were analyzed using FlowJo software v.10.7.1 (FlowJo).
Lenart K., Arcoverde Cerveira R., Hellgren F., Ols S., Sheward D.J., Kim C., Cagigi A., Gagne M., Davis B., Germosen D., Roy V., Alter G., Letscher H., Van Wassenhove J., Gros W., Gallouët A.S., Le Grand R., Kleanthous H., Guebre-Xabier M., Murrell B., Patel N., Glenn G., Smith G, & Loré K. (2024). Three immunizations with Novavax’s protein vaccines increase antibody breadth and provide durable protection from SARS-CoV-2. NPJ Vaccines, 9, 17.
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5

Comprehensive T Cell Phenotyping

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A multi-color flow cytometry panel was designed to assess T cell phenotype markers. At select time points during expansion, T cells were stained using an antibody cocktail (CD4-BUV395, CD8-APC-H7, CD25-PE-Cy7, CD27-BUV737, CD45RA-BV650, CD45RO-PE-CF594, CD69-BV786, CD197/CCR7-PE, and CD279/PD1-BV421; BD Biosciences) diluted in staining solution (1:1 mixture of Brilliant Stain Buffer Plus [BD Biosciences, #566385] and PBS with 1% FBS, 10 mM HEPES, and 2 mM EDTA) for 30 min at room temperature, centrifuged, re-suspended in fixation solution (1% formaldehyde in PBS), and immediately analyzed.
Bloemberg D., Nguyen T., MacLean S., Zafer A., Gadoury C., Gurnani K., Chattopadhyay A., Ash J., Lippens J., Harcus D., Pagé M., Fortin A., Pon R.A., Gilbert R., Marcil A., Weeratna R.D, & McComb S. (2020). A High-Throughput Method for Characterizing Novel Chimeric Antigen Receptors in Jurkat Cells. Molecular Therapy. Methods & Clinical Development, 16, 238-254.
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6

Immunophenotyping of Severe COVID-19 PBMC

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Cryopreserved PBMC from patients hospitalized because of COVID-19 severe interstitial pneumonia (severe) and OTD subjects were thawed, washed and rested for 30 min in complete RPMI-1640 medium supplemented with 10% FCS, 2mM L-glutamine and antibiotic antimycotic solution (100 U/ml penicillin, 0.1 μg/ml streptomycin, 0.25 μg/ml amphotericin B (Sigma- Aldrich, St. Louis, MO, USA) (Complete Medium). Subsequently, PBMC were washed and stained for flow cytometric analysis using the following fluorochrome conjugated antibodies: CD8 BV421, CCR7 BV510, PD1 PE-CF594, CD45RA BV650, CD56 BV786, TIM-3 BB515, CD69 PE, CD4 BB700, CD3 APC-H7 (BD Biosciences, San Diego, CA, USA). After fixation and permeabilization (Fixation/Permeabilization Solution Kit; BD Biosciences), cells were stained with anti-Granzyme B Alexa 647 (BD Biosciences). To detect circulating T follicular helper (TFH) cells 1×106 PBMC were stained with the following antibodies: CD3 BV510, CD4 BB700, CD8 BV786, PD1 PE and CXCR5 BV421. The following antibodies were used to identify plasma cells: CD19 BV605, CD27 BB515, CD38 BV421and CD138 BV480. Flow cytometry was performed with a 12-color Celesta (BD Biosciences) instrument and data analyzed with FlowJo 10 software.
Mele D., Calastri A., Maiorano E., Cerino A., Sachs M., Oliviero B., Mantovani S., Baldanti F., Bruno R., Benazzo M., Grifoni A., Sette A., Mondelli M.U, & Varchetta S. (2021). High Frequencies of Functional Virus-Specific CD4+ T Cells in SARS-CoV-2 Subjects With Olfactory and Taste Disorders. Frontiers in Immunology, 12, 748881.
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7

Multiparametric Immune Cell Profiling

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All antibodies are from BioLegend (San Diego) unless otherwise noted: CD16-APC-fire750, clone: 3G8; CD56-AF647, clone: NCAM; CD45RA-BV650, clone: HI100; CCR7-Pe/Dazzle 594, clone: G043H7; CD8-PeCy7, clone: SK1; CD45-A488, clone: SD1; CD4-AF700, clone: OKT4; CD19-BV605, clone: HIB19; CD3-BV785, clone: OKT3; HLADR-PE, clone: L243 (BD Biosciences, CA); CD14-PerCP-Cy5.5, clone: MφP9 (BD Biosciences, CA); Zombie Aqua™ Fixable Viability Kit.
Unterberg M., Ehrentraut S.F., Bracht T., Wolf A., Haberl H., von Busch A., Rump K., Ziehe D., Bazzi M., Thon P., Sitek B., Marcus K., Bayer M., Schork K., Eisenacher M., Ellger B., Oswald D., Wappler F., Defosse J., Henzler D., Köhler T., Zarbock A., Putensen C.P., Schewe J.C., Frey U.H., Anft M., Babel N., Steinmann E., Brüggemann Y., Trilling M., Schlüter A., Nowak H., Adamzik M., Rahmel T, & Koos B. (2023). Human cytomegalovirus seropositivity is associated with reduced patient survival during sepsis. Critical Care, 27, 417.
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