Flow cytometry was performed with a FACS Canto II and FACS Aria II (BD Biosciences, Mountain View, CA, USA), and the obtained results were analysed using FlowJo software (Tree Star Software, San Carlos, CA, USA). LMNCs were stained using the following monoclonal antibodies (mAbs): fluorescein isothiocyanate (FITC)-conjugated anti-DX5, APC-conjugated anti-NK1.1, APC-conjugated anti-CD120a, APC-Cy7-conjugated anti-T cell receptor (TCR)-β, PE-conjugated anti-CD69, PE-conjugated anti-CXCR3, PE-conjugated anti-natural killer group 2, member D (NKG2D), PE-conjugated anti-NK1.1, PE-conjugated anti-CD120b (all from BD Pharmingen, San Diego, CA, USA), and PE-conjugated anti-TRAIL (BioLegend, San Diego, CA, USA). Proliferative potential was tested by intracellular staining using FITC-conjugated anti- Ki-67 (Miltenyi Biotec, Auburn, CA, USA) or isotype control. Nonspecific FcγR binding of labelled mAbs was blocked by anti-CD16/32 (2.4G2) (BD Pharmingen). Dead cells were excluded from the analysis by light-scatter and/or propidium iodide staining.
Pe conjugated anti cd69
Manufactured by BD
Sourced in United States
The PE-conjugated anti-CD69 is a flow cytometry antibody that binds to the CD69 antigen, which is a type II membrane glycoprotein and a marker of early T cell activation. This antibody is conjugated with the fluorescent dye Phycoerythrin (PE), which can be detected using flow cytometry.
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Lab products found in correlation
Pe conjugated anti cd56, by BD (3 mentions)
Percp conjugated anti cd3, by BD (3 mentions)
Navios flow cytometer, by Beckman Coulter (3 mentions)
Apc cy7 conjugated anti cd3, by BD (3 mentions)
Pe cy7 conjugated anti tnf α, by BD (3 mentions)
Fitc conjugated mouse igg isotype, by BD (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Fitc conjugated anti cd25, by BD (2 mentions)
Fitc conjugated anti cd4, by BD (2 mentions)
Pe cy5 conjugated anti cd161, by BD (2 mentions)
Percp conjugated anti cd45, by BD (2 mentions)
Pe conjugated mouse igg isotype, by BD (2 mentions)
Pe conjugated anti programmed death 1 anti pd 1, by Thermo Fisher Scientific (2 mentions)
Apc conjugated anti tcr vα7, by BioLegend (2 mentions)
Fitc conjugated anti ifn γ, by BD (2 mentions)
Pe conjugated anti cd3, by BD (2 mentions)
Facsaria 2, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Anti cd16 32 2.4g2, by BD (1 mentions)
Apc conjugated anti nk1, by BD (1 mentions)
10 protocols using pe conjugated anti cd69
1
Multiparameter Flow Cytometry Analysis
2
Latency Reversal Assay Protocol
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EIF2α, p-EIF2α (Ser51), RelA, NFATc1, c-fos, ATF3, HSF1, p-HSF1 (Ser320), p24, Lamin A/C, β-actin antibodies and secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Acetylated lysine antibody, p300, CDK9 and Cyclin T1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Human FITC conjugated anti-CD25 and PE conjugated anti-CD69 antibodies were purchased from BD Biosciences (San Jose, CA, USA). PHA, poly I:C, STA-4783, thapsigargin, prostratin, PMA, ionomycin, SAHA, JQ1, dilazep, TNF-α, hemin, salubrinal, MG-132, BAY 11-7082, CsA, C646 and EX527 were from Sigma-Aldrich (St. Louis, MO, USA). Resveratrol, parthenolide (PTN), Ver-155008, KRIBB11, 17-DMAG and NVP-AUY922 were from Merck Calbiochem (Darmstadt, Germany). PEZ-HSF1 was purchased from ViGene Bioscience Inc (MD, USA). NL4-3E-R-luc plasmid, J-Lat 10.613 (link), U141 (link) and ACH242 (link) cell lines were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program.
J-Lat 10.6, U1 and ACH2 cell lines were maintained in RPMI1640 (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco) at 37 °C with 5% CO2. High temperature treatment (39.5 °C) was operated by putting cells in another incubator which temperature was adjusted to 39.5 °C.
J-Lat 10.6, U1 and ACH2 cell lines were maintained in RPMI1640 (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco) at 37 °C with 5% CO2. High temperature treatment (39.5 °C) was operated by putting cells in another incubator which temperature was adjusted to 39.5 °C.
3
Apoptosis Induction and Detection Reagents
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The following chemicals were obtained from Sigma Aldrich (USA): Glutathione (GSH), L-cysteine, D-cysteine, N-acetylcysteine (NAC), L-Buthionine-(S,R)-sulfoximine (BSO), monochlorobimane (MCB) and dihydroethidium (DHE). Monoclonal antibody (mAb) against CD3 (clone OKT3) was purified from hybridoma (ATCC) culture supernatants. Lymphoprep was from Axis-Shield PoCAS (Norway) while RPMI 1640 and FCS were from Gibco (UK). FITC-conjugated anti-CD25 and PE-conjugated anti-CD69 were acquired from BD Pharmingen (UK). The 5-bromo-2'-deoxyuridine (BrdU) labelling kit was obtained from Roche (Switzerland). Rabbit antibodies to caspase-3, mouse antibodies to β–actin and goat antibodies to caspase-8 were all from Santa Cruz Biotechnology (USA). All secondary HRP-conjugated antibodies were purchased from Dako (UK). Benzyloxycarbonyl-phenylalanine-alanine-fluoromethylketone (z-FA-FMK), benzyloxycarbonyl-tyrosine-valine-alanine-aspartic acid- fluoromethylketone (z-YVAD-FMK), benzyloxycarbonyl-valine-arginine-proline-DL-arginine-fluoromethylketone (z-VRPR-FMK) and benzyloxycarbonyl-phenylalanine-alanine-diazomethylketone (z-FA-DMK) were purchased from Bachem (Switzerland). Benzyloxycarbonyl-phenylalanine-alanine-chloromethylketone (z-FA-CMK) and biotinylated-phenylalanine-alanine-fluoromethylketone (b-FA-FMK) were from MP Biomedicals (USA).
4
Activated T Cell Profiling Post-CS Exposure
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Immediately after CS exposure and lung removal, lung tissues were digested with collagenase and DNase (Sigma, St. Louis, MO, USA) to prepare single-cell suspensions by passing the dissociated tissue through a 70-μm cell strainer (BD Falcon). The suspensions of cells were stained in PBS containing 2% FBS with the following antibodies: FITC-conjugated anti-CD4, PE-Cy7-conjugated anti-CD3, APC-Cy7-conjugated anti-CD45, PE-conjugated anti-CD69, and APC-conjugated anti-CD8 antibodies (BD Bioscience). The cells were gated on FSC (forward scatter) and SSC (side scatter). The cells were then gated on SSC versus CD45 followed by SSC versus CD3 in order to gate on all T cells. The cells were further gated to obtain the percentage of CD4+ T and CD8+ T cells. Stained cells were examined on a FACS Canto flow cytometer (BD Bioscience, San Jose, CA, USA) and analyzed using FlowJo software (TreeStar, Ashland, OR, USA). The percentages of activated CD4+ T cells and activated CD8+ T cells were determined based on double positive cells (CD69+ CD4+ or CD69+ CD8+ cells) among total CD4+ or CD8+ T cells.
5
Multicolor Flow Cytometry Immunophenotyping
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Blood samples were immunostained with the following panel of antibodies: APC-conjugated anti CD3, FITC-conjugated anti CD4, PE-conjugated anti CD69, PECy5-conjugated anti CD8, PE-conjugated anti CD28, PerCP-conjugated anti CD3, FITC-conjugated anti CD16, PE-conjugated anti CD56, PerCP-conjugated anti CD19, FITC-conjugated anti CD25, PE-conjugated anti Foxp3 and APC-conjugated anti CD4 (all reagents from BD Pharmingen, San Diego, USA). Optimal antibody concentrations were previously defined by titration. For intracellular Foxp3 staining, samples were first stained for CD4 and CD25, then fixed and permeabilized with human Foxp3 buffer set (BD Pharmingen, San Diego, USA) according to manufacturer´s protocols. Briefly, cells were washed twice with permeabilization buffer and then incubated with anti-human Foxp3 at room temperature for 30 min in the dark, before being resuspended in PBS and analyzed.
Flow cytometry data was collected on a FACSCalibur flow cytometer equipped with two lasers (Becton–Dickinson, Oxford, UK). For data acquisition and analysis CellQuestPro software (Becton–Dickinson) was used.
Flow cytometry data was collected on a FACSCalibur flow cytometer equipped with two lasers (Becton–Dickinson, Oxford, UK). For data acquisition and analysis CellQuestPro software (Becton–Dickinson) was used.
6
Multiparameter Immune Cell Profiling
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FITC-conjugated anti-Thy1.1, PE-conjugated anti-FasL, PE-conjugated anti-CD69, PE-conjugated anti-CD4, APC-conjugated anti-CD8 mAbs, and PE-conjugated anti-CD107a, corresponding isotype controls and 7-aminoactinomycin D (7-AAD) were purchased from BD Biosciences. After 30 min incubation, the stained cells were analyzed on a BD FACS Calibur (Becton Dickinson) and results were analyzed with FlowJo software. Results are illustrated as percentage of positive cells for each molecule.
7
Characterization of Monocyte Phenotype
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To study surface molecules expression, cells were washed with PBS, removed with a cell scraper, and labeled with human FITC-conjugated anti-CD14 (BioLegend, USA) or PE-conjugated anti-CD14 (BD Pharmingen, USA) and PE-conjugated anti-HLA-DR (BD Pharmingen, USA), PE-conjugated anti-CD86 (BD Pharmingen, USA) or FITC-conjugated anti-CD80 (BD Pharmingen, USA), and FITC-conjugated anti-CD40 (BD Pharmingen, USA) or PE-conjugated anti-CD69 (BD Pharmingen, USA) for 30 minutes at 4°C.
Cells were then washed twice with PBS + FCS 5% (200 ×g, 7 minutes) and resuspended in the same solution in the cold. Fluorescence measurements were performed using a FACScan or a FACSCalibur flow cytometer (Beckton, Dickinson and Company, USA). Fluorescence signals for no less than 10,000 cells were recorded in the monocyte gate, which was determined according to cell size and complexity parameters. Using these parameters, approximately 98% of the cells in this gate were CD14+. Data analyses were performed using Summit v4.3 software (Dako, USA).
Cells were then washed twice with PBS + FCS 5% (200 ×g, 7 minutes) and resuspended in the same solution in the cold. Fluorescence measurements were performed using a FACScan or a FACSCalibur flow cytometer (Beckton, Dickinson and Company, USA). Fluorescence signals for no less than 10,000 cells were recorded in the monocyte gate, which was determined according to cell size and complexity parameters. Using these parameters, approximately 98% of the cells in this gate were CD14+. Data analyses were performed using Summit v4.3 software (Dako, USA).
8
Multiparameter Flow Cytometry Analysis
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The following mAbs and reagents were used in this study: Allophycocyanin (APC)-conjugated anti-CD3, APC-conjugated anti-IL-4, APC-conjugated anti-TCRVα24-Jα18, APC-Cy7-conjugated anti-CD3, FITC-conjugated anti-CD3, FITC-conjugated anti-CD45, FITC-conjugated anti-CD56, FITC-conjugated anti-TCRγδ, FITC-conjugated anti-IFN-γ, PE-conjugated anti-TCRVα24-Jα18, PE-conjugated anti-CD45, PE-conjugated anti-CD56, PE-conjugated anti-CD69, PE-conjugated anti-IL-17A, PE-conjugated anti-lymphocyte-activation gene 3 (anti-LAG3), PE-Cy5-conjugated anti-CD161, PE-Cy7-conjugated anti-TNF-α, PerCP-conjugated anti-CD3, PerCP-conjugated anti-CD45, FITC-conjugated mouse IgG isotype, PE-conjugated mouse IgG isotype and PE-Cy7-conjugated mouse IgG isotype control (all from Becton Dickinson, San Diego, CA, USA); PE-conjugated anti-programmed death-1 (anti-PD-1; eBioscience, San Diego, CA, USA) and APC-conjugated anti-TCR Vα7.2 (BioLegend, San Diego, CA, USA). Cells were stained with combinations of appropriate mAbs for 20 min at 4°C. Stained cells were analyzed on a Navios flow cytometer using Kaluza software (version 1.5a; Beckman Coulter, Brea, CA, USA).
9
Multiparameter Flow Cytometry Immunophenotyping
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This study used the following mAbs and reagents: Allophycocyanin (APC)-Cy7-conjugated anti-CD3, APC-conjugated anti-CD3, APC-conjugated anti-CD69, and fluorescein isothiocyanate (FITC)-conjugated anti-CD45, FITC-conjugated anti-CD56, FITC-conjugated anti-CD107a, FITC-conjugated anti-IFN-γ, phycoerythrin (PE)-conjugated anti-CD3, PE-conjugated anti-CD56, PE-conjugated anti-CD69, PE-Cy7-conjugated anti-TNF-α, PerCP-conjugated anti-CD3, PerCP-conjugated anti-CD45, FITC-conjugated mouse IgG isotype and PE-conjugated mouse IgG isotype control (all from Becton Dickinson, San Diego, CA). Cells were stained with combinations of appropriate mAb for 20 minutes at 4°C. Stained cells were analyzed on a Navios flow cytometer using Kaluza software (version 1.1; Beckman Coulter, Brea, CA).
10
Immunophenotyping of T Cell Subsets
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The following mAbs and reagents were used in this study: Allophycocyanin (APC)-Cy7-conjugated anti-CD3, phycoerythrin (PE)-Cy5-conjugated anti-CD161 and fluorescein isothiocyanate (FITC)-conjugated anti-TCR γδ, FITC-conjugated anti-CD3, FITC-conjugated anti-IFN-γ, FITC-conjugated annexin V, PE-conjugated anti-CD3, PE-conjugated anti-IL-17, PE-Cy7-conjugated anti-TNF-α, PE-conjugated anti-CD69, FITC-conjugated mouse IgG isotype, PE-conjugated mouse IgG isotype and PE-Cy7-conjugated mouse IgG isotype control (all from Becton Dickinson, San Diego, CA); PE-conjugated anti-programmed death-1 (anti-PD-1; eBioscience, San Diego, CA) and APC-conjugated anti-TCR Vα7.2 (BioLegend, San Diego, CA). Cells were stained with combinations of appropriate mAbs for 20 minutes at 4°C. Stained cells were analyzed on a Navios flow cytometer using Kaluza software (version 1.1; Beckman Coulter, Brea, CA).
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