Flow cytometry was performed using a NovoCyte cytometer (ACEA, Agilent, Santa Clara, CA, USA). The following antibodies were purchased from Biolegend (San Diego, CA, USA): CD3-FITC (clone OKT3, RRID: AB_571907), CD3-APC(clone OKT3, RRID: AB_1937212), CD107a-PE-Cy7(clone H4A3, RRID: AB_11147955), CD70-PE-Cy7 (clone 113-16, RRID: AB_2687254), PE anti-Streptavidin (clone 3A20.2, RRID: AB_2571915), CD34-PE (clone 581, RRID: AB_1731862), CD33-FITC (clone HIM3-4, RRID: AB_314344), CD123-APC (clone 6H6, RRID: AB_439779), CD13-PerCP/Cyanine5.5 (clone WM15, RRID: AB_2561921), CD117-PE/Dazzle™594 (clone 2B8, RRID: AB_2564055), CD38-Alexa Fluor®700 (clone HB-7, RRID: AB_2566424), CD45-Pacific blue (clone HI-30, RRID: AB_493655). Biotinlyted CD70 protein (cat#CDL-H82D7, AcroBiosystems) was purchased from AcroBiosystems (Beijing, China), anti-mouse FMC63 (clone R19M, RRID: AB_2857947, BioSwan Laboratories) antibody was purchased from BioSwan Laboratories (Shanghai, China).
Cd3 fitc clone okt3
Manufactured by BioLegend
Sourced in United States
CD3-FITC (clone OKT3) is a fluorochrome-conjugated monoclonal antibody that binds to the CD3 antigen, which is a component of the T cell receptor complex. This product can be used for the identification and enumeration of T cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Fc receptor blocking agent, by Miltenyi Biotec (2 mentions)
Percp cy5.5 cd14 clone hcd14, by BioLegend (2 mentions)
Cd45 pacific blue clone hi30, by BioLegend (1 mentions)
Cd123 apc clone 6h6, by BioLegend (1 mentions)
Cd3 apc clone okt3, by BioLegend (1 mentions)
Cd107a pecy7 clone h4a3, by BioLegend (1 mentions)
Cd69 pe clone fn50, by BD (1 mentions)
Live dead fixable violet, by Thermo Fisher Scientific (1 mentions)
7 aad, by BioLegend (1 mentions)
Cd107a pe clone h4a3, by BD (1 mentions)
Facsfortessa 2, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Cytometer facs canto 2, by BD (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Apc cd69 clone fn50, by BioLegend (1 mentions)
Live dead fixable aqua dead cell stain, by Thermo Fisher Scientific (1 mentions)
Software, by FlowJo (1 mentions)
Facsverse, by BD (1 mentions)
Fix perm buffer, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Dojindo Laboratories (1 mentions)
5 protocols using cd3 fitc clone okt3
1
Flow Cytometry Immunophenotyping Protocol
2
Multiparametric Flow Cytometry of Immune Cell Activation
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Antibodies including CD3-FITC (clone OKT3; 1:150, BioLegend, San Diego, CA, USA), CD56 PE/Cy7 (clone NCAM1; 1:50, BioLegend), CD69-PE (clone FN50; 1:60, BD Biosciences, Heidelberg, Germany), CD107a-PE (clone H4A3; 1:25, BD Biosciences) and the corresponding isotype controls were obtained from BioLegend. 7-AAD (BioLegend) staining (1:200) or LIVE/DEAD™ Fixable Violet (1:1000, Thermo Fisher Scientific, Waltham, MA, USA) were used to identify non-viable cells within flow cytometric samples. For flow cytometric assays, 30,000 target cells were incubated in 96-well plates with 65,000 PBMCs, Rituximab at 2.5 µg/mL and various tyrosine kinase inhibitors at the indicated concentrations. After 4 and 24 h, flow cytometric analyses were performed to determine CD107a and CD69 expression, respectively. NK cells were identified as CD56+ CD3+, activated subsets as CD69+. CD107a expression indicated degranulation. Detailed gating strategies are provided in Supplementary Figure S1 . All samples were analyzed using the BD FACS Canto II or BD FACS Fortessa II (BD Biosciences, Heidelberg, Germany). Data analysis was performed using FlowJo V10 software (BD Bioscience).
3
Phenotypic Analysis of PBMCs
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Peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation with Histopaque®-1077 (Sigma) and immediately subjected to flow cytometry analysis. All experiments were performed using fresh cells. PBMCs were stained with the following fluorochrome-conjugated monoclonal antibodies: CD3-FITC (clone OKT3), CD8-Alexa 700 (clone Hit88a), CD45RA-PeCy5 (clone HI100), CCR7-PECy7, (clone G043H7), TIM3-PE (clone F38-2E2), PD1-APC (clone EH12.2H7); all antibodies from Biolegend. Data acquisition was performed with cytometer BD FACScantoII, the data were processed with FACS Diva Version 6.1.3. Gating strategy can be seen in Online Resource 1.
4
Multiparameter Flow Cytometry of PBMCs
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Multiparameter flow cytometric analysis was performed on PBMCs. Briefly, cells were incubated with Fc receptor blocking agent (Miltenyi Biotec, Bergisch Gladbach, Germany) and stained with monoclonal antibodies for 20 min at 4 °C in a darkened room. CD3 and CD14 immunophenotypic markers were used to define T lymphocytes and monocytes. Each population was also evaluated for CD142 (tissue factor; TF), and PD-L1 expression. The following monoclonal antibodies were used (all from BioLegend, San Diego, CA, USA): FITC-CD3 clone OKT3, PerCP/Cy5.5-CD14 clone HCD14, APC-CD69 clone FN50, PE-CD142 clone NY2, PE/Cy7-HLA-DR clone L243, Brilliant Violet 421-PD-L1 clone 29E.2A3 were used. Matched isotype controls were used for each antibody to establish the gates. Live cells were discriminated by means of LIVE/DEAD Fixable Aqua Dead Cell Stain (Thermo Fisher Scientific, Waltham, MA, USA) and dead cells were excluded from all analyses. All flow cytometric analyses were performed using a BD FACSVerse™ (BD, Franklin Lakes, NJ, USA). Data were analyzed using FlowJo software (FlowJo LLC, Ashland, OR, USA).
5
Multiparametric Immunofluorescence Profiling of PBMCs
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Immunofluorescence staining was performed on PBMCs. Briefly, cells were incubated with Fc receptor blocking agent (Miltenyi Biotec, Bergisch Gladbach, Germany) and stained with monoclonal antibodies for 20 min at 4 °C in a darkened room. The following monoclonal antibodies were used (all from BioLegend, San Diego, CA, USA): FITC-CD3 clone OKT3, PerCP/Cy5.5-CD14 clone HCD14, APC-CD69 clone FN50, PE-CD142 clone NY2. After fixation of stained cells using Fix/Perm buffer (Thermo Fisher Scientific, Waltham, MA, USA), the suspension of fixed cells was immobilized onto glass slides by cytospin. Nuclei were counter stained with 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) (DOJINDO, Kumamoto, Japan) in water, and whole sections were mounted in ProLong Diamond (Thermo Fisher Scientific, Waltham, MA, USA). Slides were observed with a confocal fluorescence microscope (FV3000, Olympus, Tokyo, Japan).
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