Cells were harvested after 24 h, 48 h, and 1 week. The expression of surface markers was analyzed by flow cytometry using anti-CD80-FITC, anti-CD70-PE, anti-CD40-PE and their corresponding isotype controls, and anti-CD56-FITC, anti-CD3-APC-Cy7 or anti-CD3-V500, anti-CD69-PE, anti-CD25-BV421 or anti-CD25-PE, and anti-CD54-APC or anti-CD54-PE (all from BD Biosciences) as recently described.33 (link) Immunofluorescence was measured using a FACS Canto II (BD Biosciences), data were acquired with FACSDiva software (BD Biosciences) and evaluated with FCS Express software, version 5 (DeNovo Software). An average of approximately 6500 NK cells per measurement was acquired, with a minimum of 500 and a maximum of 23,000 cells.
Anti cd25 bv421
Manufactured by BD
Sourced in United States
Anti-CD25-BV421 is a fluorescent-labeled antibody that binds to the CD25 cell surface marker. It is designed for use in flow cytometry applications to identify and analyze cells expressing CD25.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd25 pe, by BD (3 mentions)
Anti cd3 v500, by BD (3 mentions)
Anti cd4 pe cy7, by BD (3 mentions)
Anti cd56 fitc, by BD (2 mentions)
Anti cd3 apc cy7, by BD (2 mentions)
Anti cd69 pe, by BD (2 mentions)
Anti cd54 apc, by BD (2 mentions)
Anti cd54 pe, by BD (2 mentions)
Anti cd8 apc, by BD (2 mentions)
Anti foxp3 pe, by BD (2 mentions)
Facscanto 2, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Anti cd80 fitc, by BD (1 mentions)
Anti cd40 pe, by BD (1 mentions)
Fcs express software, by De Novo Software (1 mentions)
Anti cd70 pe, by BD (1 mentions)
Anti cd3 pe cy7, by BD (1 mentions)
Anti cd14 apc, by BD (1 mentions)
Anti cd56 pe, by BD (1 mentions)
Anti cd8 apc h7, by BD (1 mentions)
10 protocols using anti cd25 bv421
1
Analysis of NK Cell Surface Markers
2
Immune Cell Analysis of COVID-19 Vaccinated Donors
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Human PBMCs collected from likely COVID-19-vaccinated donors between February 2022 and February 2023 were isolated from buffy coats by means of Ficoll density gradient centrifugation and infected for 24 h with 5 MOI of Prime-2-CoV_Beta (SPEk103) or left untreated. Cells were collected by scraping, washed in PBS and incubated for 30 min at 4 °C with the following antibody cocktail in PBS: anti-CD14 APC (BD Biosciences, San Jose, CA, USA, 561383), anti-CD4 BV605 (BD Biosciences, 562843), anti-CD8 APC-H7 (BD Biosciences, 561423), anti-CD19 BV786 (BD Biosciences, 563325), anti-CD3 PE-Cy7 (BD Biosciences, 557749), anti-CD56 PE (BD Biosciences, 556647), anti-CD25 BV421 (BD Biosciences, 567485), LIVE/DEAD™ Fixable Aqua stain (Invitrogen, Waltham, MA, USA, L34957). After three washes in PBS, cells were fixed in 4% formaldehyde for 20 min, washed and finally resuspended in FACS buffer. Data were acquired with an Attune NxT cytometer (Thermo Fisher Scientific). Data analysis was performed using Kaluza software (version 2.1, Beckman Coulter, Brea, CA, USA).
3
Quantification of Th1, Treg, and Th17 Cells by Flow Cytometry
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Mouse spleens were collected, ground and filtered through a sieve to obtain single cell suspensions. To detect Th1, Treg and Th17, cells were stimulated with phorbol myristate acetate (PMA)/ionomycin mixture and GolgiPlug (BD Biosciences) for 4h. Anti-CD4-FITC (BD Biosciences, 553046), Anti-CD3e APC-Cy7 (BD Biosciences, 557596) and Anti-CD25-BV421 (BD Biosciences, 607180) were used to stain the surface markers. After washing, cells were fixed and permeated using the Fixation/Permeabilization Kits (eBioscience). Anti-Foxp3-Alexa 647 (BD Biosciences, 560401), Anti-IFNG-PE-Cy7 (BD Biosciences, 557649), Anti-IL17A-PE (BD Biosciences, 559502) antibody was used for staining of intracellular markers. Finally, stained cells were detected by flow cytometry (FACSAriaIII, BD Biosciences) and data were analyzed with Flowjo software.
4
Flow Cytometric Analysis of Immune Cell Subsets
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Cultured cells were stained for flow cytometry analysis of various surface receptors using combinations of the following antibodies: anti-CD3-BV786 or BV605, anti-CD4-PeCy7 or PerCP-Cy5.5, anti-CD8-FITC or APC-H7, anti-CD25-BV421, anti-CD25-PE and mouse IgG2aκ-PE isotype control (all from BD Biosciences, San Jose, CA, USA). Live/Dead Fixable Aqua Dead Cell Stain Kit (Molecular Probes, Invitrogen) or 7-Aminoactinomycin D (7AAD, BD Biosciences) were used to exclude non-viable cells according to the manufacturers’ protocols. For analysis of apoptosis, cells were labelled with Annexin V diluted in Annexin V Binding Buffer (BD Biosciences). Samples were collected with a LSRII or FACSFortessa X20 flow cytometer (BD Biosciences) using DIVA software (BD Biosciences) and analyzed using FlowJo software (Tree Star Inc., Ashland, KY, USA). Gates were set using Fluorescence Minus One (FMO) or isotype control.
5
Flow Cytometric Characterization of T cells
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PBMCs were stained with the Acqua Live/Dead Stain (Invitrogen, Carlsbad, CA, USA) for 20 min at room temperature. Then, the following panel of anti-human fluorochrome-conjugated antibodies were used for flow cytometric characterization of T cells: anti-CD3-PerCP (BD Biosciences, San Diego, California, USA), anti-CD4-APC (BD Biosciences), anti-CD4-PE-cy7 (BD Biosciences), anti-CD25-BV421 (BD Biosciences), anti-CD127-FITC (eBioscience, Waltham, MA, USA), anti-CD45RO-BV510 (eBioscience), CD39-PE (eBioscience). After incubation for 30 min at 4°C in the dark, the cells were washed with FACS buffer and fixed. For intracellular staining, cells were fixed and permeabilized using Foxp3 staining kit (eBioscience) then stained with anti-Foxp3-APC (BD Biosciences). Data were acquired on a BD-FACSCanto (BD Biosciences) and analyzed using FlowJo software V10 (Tree star lnc., Ashland, OR, USA).
6
Treg Cell Isolation and Killing Assay
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Treg cells were obtained from two sources: (i) PBMC from two healthy donors were purified from blood in‐house using Ficoll–Hypaque density gradient centrifugation (Robbins Scientific) and Treg cells were extracted using a CD4+ CD25+ CD127dim/− Regulatory T Cell Isolation Kit II (Miltenyi Biotech Ltd.). Treg cell purity was assessed by labelling cells with anti‐human antibodies: anti‐CD3‐FITC, anti‐CD8‐APC, anti‐CD4‐PE‐Cy7, anti‐CD25‐BV‐421 and anti‐FoxP3‐PE (BD Bioscience) and subsequent FACS analysis (Intellicyt iQplus platform and ForeCyt software analysis), (ii) Treg cells were sourced from Tissue Solutions (Glasgow, UK). CD8 T were isolated by negative selection using CD8+ T Cell Isolation kit (Miltenyi Biotech) or provided by Tissue Solutions. A Treg cell killing suppression assay was carried out using the IncuCyte ZOOM‐Platform (EssenBioScience, Ann Arbor, MI). Target cells (Mel526) were plated at 1·5 × 104 cells per well of a 96‐well plate in R10 and incubated overnight at 37°C in 5% CO2. Effector CD8+ T cells and Treg cells were added at 6 × 104 cells per well (at 1 : 1 CD8+ T cell/Treg cell ratio). IMCgp100 was added at 100 pm ; 5 μm NucView (EssenBioScience) was added into each well to allow detection of apoptosis.
7
Flow Cytometric Analysis of Immune Cell Activation
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Cells were harvested either 2, 4, 6, and 24 h after electroporation or after 48 h of co-culture. The expression of surface markers was determined via flow cytometry using anti-CD215-PE (anti-human IL-15Rα, clone JM7A4) (Biolegend, San Diego, CA, USA) and IgG2b-PE isotype control, anti-CD56-FITC, anti-CD3-APC-Cy7 or anti-CD3-V500, anti-CD69-PE, anti-CD25-BV421 or anti-CD25-PE, and anti-CD54-APC or anti-CD54-PE (all from BD Biosciences, Heidelberg, Germany) as described [39 (link)]. A FACS Canto II flow cytometer (BD Biosciences) and FACSDiva software [40 ] were used to measure immunofluorescence and acquire data, which was evaluated with FCS Express software [41 ].
8
Immunological Evaluation of Vaccine Adjuvants
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Hematoxylin & eosin, periodic acid–Schiff (PAS) and Masson’s trichrome (TRI) dyes were from Merck Millipore, USA. Alum adjuvant (Pierce), RevertAid H Minus Reverse Transcriptase, and Rapid DNA Ligation Kit were from Thermo Fisher Scientific, USA. Limulus amoebocyte lysate assay kit was from Biolasco, Taiwan. Adenosine assay kit was from Cell Biolabs, USA. Tetanus toxoid (TT) was from Biofarma, Indonesia. Anti-CD16/32 (FcX), anti-CD3-FITC, anti-CD4-PerCP and anti-FoxP3-Alexa Flour®647 were from BioLegend, USA. Anti-CD25-BV421 and anti-CD45RA-PE were from BD Biosciences, USA. IDO1 ELISA kit was from LifeSpan Biosciences, USA. Phosphatidylcholine was from Lipoid-AG, Switzerland. Cholesterol was from Sigma-Aldrich, Germany. Dimethyldioctadecyl-ammoniumbromide (DDAB) was from Honeywell Research Chemicals, USA.
9
Multiparametric Phenotyping of PBMCs
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PMBCs were thawed in Benzonase Nuclease (Sigma-Aldrich)/X-VIVO medium (Lonza), washed with RPMI medium and stained with the following fluorochrome-conjugated antibodies: anti-CD14 – PerCP-Cy5.5 (BD, clone MΦP9), anti-HLA-DR – V500 (BD, clone G46–6), anti-PD-L1 – PE-Cy7 (BD, clone MIH1), anti-CD73 – BV605 (Biolegend, clone AD2), anti-CD3 – V500 (BD, clone SP34–2), anti-CD4 – APC-Cy7 (BD, clone RPA-T4), anti-CD8 – APC (BD, clone RPA-T8), anti-CD69 – PE-Cy7 (BD, clone FN50), anti-PD-1 – PerCP-Cy5.5 (BD, clone EH12.1) and anti-CD25 – BV421 (BD, clone M-A251). Dead cells were discriminated with the Fixable Viability Stain A×700(BD). To reduce unspecific antibody binding, FcR Blocking Reagent (Miltany) was added. Analysis of the intracellular marker FOXP3 – Alexa 488 (BD, clone 259D/C7) were conducted using the FOXP3/Transcription Factor Fixation/Permeabilization kit (ThermoFisher). Acquisition was performed by 10-color flow cytometry using BD FACSLyric with FACSuite software (BD Biosciences). FlowJo V 10 software (BD Biosciences) was used to analyze at least 106 events. Positively stained cells were gated according to the fluorescence minus one (FMO) control.
10
Comprehensive Immune Cell Profiling
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To evaluate the relative proportions of lymphocyte populations in peripheral blood, the following panel of fluorochrome conjugated antibodies was used: anti CD45 BUV 395, anti CD3 APC R700, anti CD19 APC, anti CD16 PE-Cy7, anti CD56 BB515, anti CD4 BUV 7373, anti CD8 BV 785, anti CD25 BV 421, anti CD127 BB700, anti CD45RA BV605, anti CD197 BV711, anti FoxP3 PE, anti RORγT BV650 (all from BD Biosciences, Milan, Italy). Blood samples were stained with anti membrane associated molecules and, after incubation, erythrocytes were lysed using FACS lysing solution, according to manufacturer’s instruction. To analyze the expression of transcription factors (FoxP3 and RORγT), samples were fixed and permeabilized using Transcription Facytors Buffer Set (BD Bioscience), according to standard protocol. Samples were acquired and analyzed on a LSR Fortessa X-20 (Becton Dickinson), using FACS DIVA v8.0.2 software.
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