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10 protocols using cd45ra bv605

1

Comprehensive Immunophenotyping by Flow Cytometry

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All cells were stained for viability using the Zombie Fixable Viability Kit (Biolegend), incubated with anti-CD16/32 Fc-block (BioXcell), and stained with the indicated antibodies: CD19 APC, CD3 APC-H7 or BV450, CD4 AF700, CD8 V500, CCR7 PE-CF594, CD69 BV785, CCR5 PE, CD103 FITC, (BD Biosciences) CD38 PE-Cy7, CD11c BV711, CD14 BV650, CD45RA BV605 (Biolegend). Stained samples were run on an LSRII flow cytometer, data acquired using FACS DIVA software (BD Biosciences) and analyzed using FlowJo software (TreeStar, Inc., Ashland, Oregon).
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2

T Cell Phenotyping and Flow Cytometry

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For protein expression analysis following proliferation, CD8 or CD4 T cells were isolated and stimulated as described above. On day 4 of the proliferation assay cells were collected and stained with the following antibodies for surface protein expression: CD3 APC-Cy7 (Biolegend), CD4 AF-700 (Biolegend), CD8 PE-Cy7 (Biolegend), CD45RA BV-605 (Biolegend), CCR7 AF-488 (Biolegend) or CCR7 PE-Dazzle594 (Biolegend), and CD19 PerCP-Cy5.5 (Biolegend). Dead cells were excluded from the analysis by using Zombie-UV (Biolegend). Doublets and double positive CD4/CD8 cells were removed through sequential gating. Flow cytometry acquisition was done using the BD LSRII with BD FACSDiva and Cyteck Aurora. Data was analyzed by FlowJo 10.1r7 and GraphPad Prism 9.
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3

Multi-Dimensional Immune Profiling

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For protein expression analysis following isolation cells were collected and stained with the following antibodies for surface protein expression: CD3 APC-Cy7 (BioLegend), CD4 AF-700 (BioLegend), CD8 PE-Cy7 (BioLegend), CD45RA BV-605 (BioLegend), CD27 AF-488 (BioLegend), CD8 BUV-496 (BD Biosciences), CD3 BUV-805 (BD Biosciences), CD4 BV-750 (BD Biosciences), CD45RA BUV-395 (BD Biosciences), and CCR7 BV-786 (BD Biosciences). Dead cells were excluded from the analysis by using Zombie-UV (Biolegend). Doublets and double-positive CD4/CD8 cells were removed through sequential gating. Flow cytometry acquisition was done using the BD LSRII with BD FACSDiva. Data was analyzed by FlowJo 10.1r7 and GraphPad Prism 9.
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4

Multiparametric Flow Cytometry for Immune Profiling

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Surface marker expression on DCs was assessed via flow cytometry by staining the cells with α-HLA-DR APC, Fixable Viability Dye eFluor506, α-CD1a BV421, α-CD86 PE (eBioscience), α-CD14 PerCP-Cy5.5 (BD Bioscience) and α-ILT3 PerCP-Cy5.5 (Biolegend). Samples were subsequently acquired using a FACS Canto II flow cytometer (BD Biosciences). The shown MFI data were collected after exclusion of doublets and dead cells.
Surface marker expression on PBMCs was assessed via flow cytometry using a 13-color flow-cytometry panel generated by using the Optimized Multicolor Immunofluorescence Panel-030 (OMIP-030)34 as a reference: CD4 BV510, CD127 AlexaFluor 700, CD8a PE-Cy5, CD3 PerCP-Cy5.5, CD45RA BV605, CD25 PE, CD20 APC-Cy7, CD194 PE-Cy7, CD197 PE/Dazzle594 (Biolegend), CCR10 APC (BD Biosciences), Ki-67 BV650, CD196 BB151, CD183 BV421 (BD Horizon) Fixable Viability Dye APC-Cy7 (eBioscience). Samples were acquired using a Cytoflex S flow cytometer (Beckman Coulter) and then analyzed using the gating strategy summarized in ESI Fig. 5.
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5

Multiparameter T Cell Phenotyping

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Blood samples were drawn from patients following permission and processed at the University of Arizona Biorepository Laboratory. Peripheral blood mononuclear cells were cryopreserved for future analysis. A complete blood count was performed using an Ac-T 5diff CP machine (Beckman Coulter, Pasadena, CA). Cryopreserved PBMC (1–2x106/sample) were stained with LIVE/DEAD Fixable Dead Cell Stain-AQUA (Invitrogen) and T cell markers in various combinations.11 (link)
The following mAbs were used to differentiate T cell subsets: CD3—BV570 (BioLegend), CD4—APC (eBioscience), CD8β—ECD (Beckman Coulter), CD95—BV421 (BioLegend), CD28—PerCp/Cy5.5 (BioLegend), CCR7—FITC (BD Pharmogen), CD45RA—BV605 (BioLegend), CD57—BV570 (BioLegend), IFN-γ—APCe780 (eBioscience). Cells following various combination and incubations as described in our previous study,11 (link)
were analyzed on the BD LSR II instrument using DiVa acquisition (BDIS, Mountain View, CA) and the FlowJo analysis software (TreeStar Inc., Ashland, OR).
Twenty (20) of 37 patients in this study completed the entire cell surface analysis.
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6

Multiparametric Immune Profiling of PBMC

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Cryopreserved PBMCs were thawed and left for 1 h at 37 °C in the CO2 incubator. Subsequently the cells were collected and stained with BD Horizon™ Fixable Viability Stain 510 to identify live cells, as per manufacture protocol. The cells were then washed, and surface stained for IL-21R PE expression on, (i) CD4, CD8 and Tfh (CD4/CD45RA -/CXCR5+/PD1+); (ii) B cell CD20, B1(CD20+CD27+CD43+), Plasma blast (CD20+CD38+) and iii) monocytes CD14+HLADR+.
After staining, the cells were washed and fixed using 2% PFA. Acquisition was done on BD FACSCelesta (Becton-Dickenson, San Jose, CA). Forward and side scatters and singlets were used to gate and exclude cellular debris. The flow cytometry results were analyzed using FlowJo™ v10.8 Software (BD Life Sciences, Ashland, OR). The details of the antibodies used are as follows: Fixable Viability stain510, CD4 FITC (clone: RPA-T4) from BD Bioscience (San Jose, CA), IL-21R PE (clone: 2G1-K12), CD8 PerCP (clone: SK1), PD1 APC (clone: EH12.2H7), CXCR5 BV421 (clone: J252D4), CD45RA BV605 (clone: HI100), CD38 BV421 (clone: HB-7), CD16 AF700 (clone: B73.1), CD14 BV650 (clone: M5E2), HLADR BV605 (clone: L243), CD20 PerCP (clone: 2H7), CD27 FITC (clone: M-T271), CD43 APC (clone: CD43-10G7) from BioLegend (San Diego, CA).
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7

Multiparametric Phenotyping of Immune Cells

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Stimulated cells were stained with fluorochrome-conjugated monoclonal antibodies to detect surface and intracellular markers. Intracellular staining was performed with Foxp3 Fixation/Permeabilization buffer kit (eBioscience, San Diego, CA, USA), and Fixable Viability eFluor 780 Dye (eBioscience) was used to exclude dead cells. The following antibodies were used: CD4-Alexa Fluor 700 (OKT4, BioLegend), CD8-V500 (RPA-T8, BD Biosciences, San Jose, CA, USA), GrB-Fitc (GB11, BioLegend), IL-2-Pe (MQ1-17H12, BioLegend), IL-4-PeDaz594 (MP4-25D2, BioLegend), CD137-Pe/Cy7 (4B4-1, BioLegend), CD154-Alexa Fluor 647 (24-31, BioLegend), TNF-α-eFl450 (MAb11, eBioscience), IFN-γ-BV650 (4S.B3, BioLegend), CD3-BV785 (OKT3, BioLegend), CD19-BV605 (HIB19, BioLegend), CD45RA-BV605 (HI100, BioLegend), CCR7-PerCP/Cyanine 5.5 (G043H7, BioLegend).
For the absolute quantification of immune cell subsets, total CD19+, CD3+, CD4+, and CD8+ T cells numbers were determined in 50 μL of the peripheral blood by flow cytometry as described below by means of CytExpress software (Beckman Coulter, USA). Absolute numbers of HBs-specific T cells were determined based on relative frequencies of HBs-specific T cells and absolute numbers of total CD4+ T cells per μL of peripheral blood.
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8

Phenotypic analysis of CAR T cells

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The following reagents were used for phenotypic analysis of CAR T cells: CD2 APC (Miltenyi), CD3 PerCPCy5.5 (Biolegend), CD4 PE-Vio770 (Miltenyi), CD8 PE or FITC (Biolegend), CD19 BV605 (Biolegend), CD19 PE (Biolegend), CD45RA BV605 (Biolegend), CCR7 APC (Biolegend), CD107a FITC (BD), CD223 APC-eFluor 780 (LAG-3, eBioscience), CD279 BV421 (PD1 Biolegend), CD366 (TIM3 BV711), IFN-gamma APC, TNF-alpha BV421, IL-2 BV605 (Biolegend), Anti-Rabbit Goat F(ab')2 FITC (Jackson Immunoresearch), Anti-Rabbit IgG BV421 (Biolegend), Anti-Mouse IgG PE (Biolegend), Fixable viability dye Life technologies Aqua. Fluorescence minus one (FMO) controls were used to determine expression thresholds where required.
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9

Cytomegalovirus-specific CD8+ T cell profiling

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Blood samples were obtained from a donor with detectable NLV-specific CD8+ T cell response. Lymphocytes were enriched via Ficoll gradient and prestained with a near-IR fixable live/dead marker (Life Technologies, cat# L34976) and an APC-conjugated dextramer reagent (Immudex, cat# WB2132-APC). The cells were then stained with the following antibodies: FITC CD8A (BioLegend, cat# 300906), PerCP-Cy5.5 CCR7 (BioLegend, cat# 353220), PE CD3 (BioLegend, cat# 317308), BV605 CD45RA (BioLegend, cat# 304133).
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10

Multiparametric Flow Cytometry Profiling

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Differentiated cells were washed in PBS before being singularized using TrypLE (Thermo Fischer Scientific), passed through a 21G needle, and filtered using 30 μm sterile Cup Filcons (BD Biosciences). Cells were treated with 7AAD to exclude dead cells. Cells were stained using the following anti-human antibodies; fluorescein isothiocyanate (FITC)-conjugated CD45 (eBioscience, 11-0459-42) and CD43 (BD Biosciences, 555475), phycoerythrin-cyanine (PE-Cy7)-conjugated CD34 (BioLegend, 343516), allophycocyanin-conjugated CD38 (BioLegend, 303510), CD33 (BioLegend, 303408), CD14 (BioLegend, 325608) Phycoerythrin (PE)-conjugated CD90 (BioLegend, 328110), CD19 (BioLegend, 302208), CD7 (BD Biosciences, 332774), V450-conjugated CD45RA (BD Biosciences, 560362), PE-Cy5-CD56 (BioLegend, 304608), BV605-CD45RA (BioLegend, 304133), and eFluor450-CD16 (eBioscience, 48-0168-42). Cells were acquired on a FACS Canto II (BD Biosciences) or sorted using a FACS Aria III (BD Biosciences). Analysis was done using FlowJo, version 9.4.10 (BD Biosciences). FACS gates are based on fluorescence minus one (FMO) controls unless stated otherwise.
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