Spleen cells were prepared as described above. The cells were surface-stained with fluoresce in isothiocyan (FITC)-conjugated anti-CD4 (BD Pharmingen, San Diego, CA, USA) and PE-conjugated anti-CD25 (eBioscience) to separate T cells. For analysis of the FoxP3, cells were fixed, permeabilized and stained according to the manufacture instruction for FoxP3 staining (PE-Cy5-conjugated anti-mouse/rat FoxP3 staining kit; eBioscience). To analyze intracelluar cytokine production, the cells were cultured for 48 h in the prescence of 2 mM monensin (BD Pharmingen) with 100 ng/ml phorbolmyristate acetate (PMA) (Alexis, Lausen, Switzerland) and 1 mM ionomycin (Alexis). After washed and blocked with Fc-blockade (CD16/32; BD Pharmingen) for 30 min, cells were directly surface-stained with PE-Cy5-conjugated anti-CD3e (BD Pharmingen) and FITC-conjugated anti-CD4. After fixed and permeabilized using the BD cytofix/cytoperm kit (BD Pharmingen), the cells were stained using PE-conjugated anti-IL-4 (BD Pharmingen) or PE-conjugated anti-IFN-γ (BD Pharmingen). Cells were analyzed (104 gated events were collected) using a FACSCalibur. Background fluorochrome was assessed using appropriate isotype and fluorochrome-conjugated control mAbs. Data collected were analyzed using FlowJo software.
Pe conjugated anti ifn γ
Manufactured by BD
Sourced in United States
The PE-conjugated anti-IFN-γ is a fluorescently-labeled antibody that binds to the interferon-gamma (IFN-γ) protein. It can be used to detect and quantify the presence of IFN-γ in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Cytofix cytoperm kit, by BD (4 mentions)
Golgistop, by BD (4 mentions)
Fitc conjugated anti cd4, by BD (3 mentions)
Perm wash, by Thermo Fisher Scientific (3 mentions)
Perm wash, by BD (3 mentions)
Pe conjugated anti il 4, by BD (2 mentions)
Ic fixation buffer, by Thermo Fisher Scientific (2 mentions)
Cytofix cytoperm, by BD (2 mentions)
Golgiplug, by BD (2 mentions)
Anti tnf α, by Thermo Fisher Scientific (2 mentions)
Pe conjugated anti cd25, by Thermo Fisher Scientific (1 mentions)
Monensin, by BD (1 mentions)
6 gingerol, by Merck Group (1 mentions)
Fluorescein isothiocyanate fitc conjugated anti cd11b, by BD (1 mentions)
Fitc conjugated anti cd8, by BD (1 mentions)
Pe conjugated anti cd4, by BD (1 mentions)
Pe conjugated anti cd8, by BD (1 mentions)
F4 80 pe, by Thermo Fisher Scientific (1 mentions)
P aeruginosa atcc 27853, by American Type Culture Collection (1 mentions)
Anti il 6, by Thermo Fisher Scientific (1 mentions)
11 protocols using pe conjugated anti ifn γ
1
Analysis of T Cell Subsets and Cytokine Production
2
Ginger Compound Modulates Immune Responses
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6-Gingerol (Sigma-Aldrich, St Louis, MO) was dissolved to 3.5 μg/ml of 50% ethanol and diluted with drinking water, as required (the final ethanol concentration was less than 0.01%). The following antibodies were purchased from BD Pharmingen, San Diego, CA: phycoerythrin- (PE-) conjugated anti-Ly6G, fluorescein isothiocyanate- (FITC-) conjugated anti-CD11b, PE-conjugated anti-CXCR2, FITC-conjugated anti-CD4, FITC-conjugated anti-CD8, PE-conjugated anti-IFN-γ, PE-conjugated anti-CD4, PE-conjugated anti-CD8, and FITC-conjugated antiannexin V antibodies. P. aeruginosa (ATCC27853) was obtained from the American Type Culture Collection (ATCC). For the M1 and M2 macrophage analysis, cells were separately stained with monoclonal antibodies specific for F4/80-PE from eBioscience; F4/80-FITC and CD206-PerCP/Cy5.5 from BioLegend, San Diego, CA; and CD80-FITC and iNOS-FITC from BD Transduction Laboratories, San Diego, CA. Bacteria were grown in brain heart infusion (BHI) media (Difco) at 37°C for 18 h, and aliquots were frozen at −80°C.
3
Profiling Cytokine Responses in S. aureus Infection
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Immune cells collected from S. aureus (1 × 107 CFUs per head)–infected mice were incubated with brefeldin A solution for 3 hours at 37°C in a round-shaped 96-well microplate. After adding BD GolgiStop, cells were stained with fixable viability dye for 30 min. For the measurement of surface expression of immune cell markers, cells were stained for 45 min after Fc blocking at 4°C. Stained cells were fixed with intracellular (IC) fixation buffer for 30 min and permed for 20 min at room temperature. Then, permed cells were stained with phycoerythrin (PE)–conjugated anti–IFN-γ (BD Biosciences, San Jose, CA, USA), anti–IL-6, anti–IL-10, anti-TNFα (Thermo Fisher Scientific, Waltham, MA, USA), and anti-CCL2 (BioLegend, San Diego, CA, USA) for 1 hour. Data were analyzed using a BD FACS Canto II flow cytometer and FlowJo analytical software (BD Biosciences, San Jose, CA, USA).
4
Detecting IFN-γ in Activated NK Cells
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To detect intracellular production of IFN-γ, PBMCs from patients and healthy donors were incubated overnight at 37°C with IL-12 (0.5 ng/ml), or IL-12 (0.5 ng/ml) and IL-18 (0.1 ng/ml) combined. Surface staining was done by incubating the cells with FITC anti-CD3, PC5 anti-CD56, FITC anti-CD14 (Beckman Coulter), FITC anti-CD20, and APC/Cy7 anti-CD16 (BD) mAbs for 30 min at 4°C. Cells were then washed, fixed, and permeabilized with BD Cytofix/Cytoperm kit (BD Biosciences Pharmingen). IFN-γ production was detected by subsequent intracellular staining with PE-conjugated anti-IFN-γ (BD Biosciences Pharmingen). After washing, the proportion of CD3− CD14− CD20− CD56+ cells expressing IFN-γ was immediately analyzed on LSR Fortessa Flow Cytometer (BD) using FACSDiva software (BD). Final analysis was done using FloJo v.10.2 (TreeStar).
5
Profiling Antigen-Specific T Cell Responses
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Splenocytes were stimulated with either 0.2 μg/mL of HLA B35 (“HPV” and “LPEP”), HLA A2 (“GLC” and “CLG”), HLA A24 (“TYG” and “PYL”), HLA B8 (“FLR” and “RAK”) restricted peptides, or gp350 OLP PepMix™ EBV, a pool of 224 peptides derived from a peptide scan (15mers with 11 aa overlap) through envelope glycoprotein GP350/GP340 (Swiss-Prot ID: P03200) of Epstein-Barr virus (HHV4) (Product Code: PM-EBV-GP350/GP340; JPT Peptide Technologies GmbH, Berlin, Germany). Splenocytes were stimulated in the presence of Golgiplug™ and Golgistop™ for 5 hours in a 37 °C humidified incubator with 6.5% CO2. After incubation, cells were washed twice, then incubated with Live/Dead™ near IR (invitrogen, cat# L34976, 1:250), FITC-conjugated anti-CD4 (BD Biosciences, cat# 553651, 1:200), and PerCP-Cy5.5 conjugated anti-CD8 (BD Biosciences, cat# 551162, 1:200). Cells were fixed and permeabilized using a BD Cytofix/Cytoperm™ kit, then incubated with PE-conjugated anti-IFNγ (BD Biosciences, cat# 554412, 1:200), PE-Cy7 conjugated anti-TNFα (BD Biosciences, cat# 557844, 1:200), and APC-conjugated anti-IL-2 PE (BD Biosciences, cat# 554429, 1:10). Cells were acquired on a BD FACSCanto™ II and data was analyzed (Supplementary Fig. S13 ) using FlowJo™ software.
6
Multiparameter Flow Cytometry Panel
7
Multiparametric Flow Cytometry for NK Cells
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Single-cell suspensions were prepared as previously described (Dou et al., 2018 (link)). For detection of NK cells and their NKG2D expression, cells were stained with FITC-conjugated anti-NK1.1 (BD Bioscience), APC-conjugated anti-CD3 (BioLegend), PE-conjugated anti-NKG2D (BioLegend) and corresponding isotype controls. For detection of NK cell-produced IFN-γ, firstly, 1.0×106 cells were activated in vitro with 2 μl Leukocyte Activation Cocktail (BD Bioscience) for 5 h at 37°C, followed by incubation with FITC-conjugated anti-NK1.1, APC-conjugated anti-CD3 for 30 min. Then, cells were permeabilized and fixed using Cytofix/Cytoperm Soln Kit (BD Bioscience) for 30 min, followed by incubation with PE-conjugated anti-IFN-γ (BD Bioscience). FACS was performed on BD FACS Calibur flow cytometer (BD Biosciences, NJ, United States) or CytoFLEX (BECKMAN COULTER, United States). Data were analyzed using FlowJo (version 10, United States). For analysis of NK cell infiltration, the lymphocyte population was gated from the general diagram of FSC-SSC. The negative control was then determined from cells stained with isotype controls. The location of CD3+ cells was determined by CD3-stained cells, and that of NK1.1+ cells was distinguished by NK1.1-stained cells. The percentage of NK1.1+CD3− cells in the population was recorded.
8
Multiparametric Flow Cytometry Analysis
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Single cell suspensions from immunised and infected mice (2 × 106 cells) were stimulated with Rv3131 (5 μg/mL) or PPD (2 μg/mL) at 37 °C for 12 h in the presence of GolgiStop (BD Bioscience). Cells were first washed with PBS, blocked with an anti-CD16/32 blocking antibody at 4 °C for 15 min and then surface stained with fluorochrome-labelled antibodies against CD3ε, CD4, CD8 and CD62L as well as the LIVE/DEADTM Fixable Aqua Dead cell kit for 30 min at 4 °C. As negative controls, cells were surface stained with the proper isotype-matched immunoglobulin (Ig). Cells were washed, fixed and permeabilised with Cytofix/Cytoperm (BD Biosciences) for 30 min at 4 °C. Cells were washed twice with Perm/Wash (BD Biosciences) and then stained intracellularly with APC-conjugated anti-TNF-α, PE-conjugated anti-IFN-γ and PE-Cy7-conjugated anti-IL-2 (BD Biosciences) for 30 min at 4 °C. Cells were washed with Perm/Wash and then fixed with IC Fixation Buffer (eBioscience). After being resuspended in PBS, cells were analysed using a flow cytometer.
9
Analyzing Th1 and Th17 Responses to RpfE Antigen
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Four weeks after the last immunization, the mice were euthanized by CO2 asphyxiation, and single-cell suspensions (1 × 106 cells) from immunized mice were re-stimulated with RpfE (10 μg/mL) at 37 °C for 12 h in the presence of both GolgiPlug and GolgiStop (BD Biosciences, Franklin Lakes, NJ, USA). PBS-washed cells were blocked with anti-CD16/32 antibody (BD Biosciences) at 4 °C for 20 min. The cells were then surface stained with Brilliant Violet (BV) 605-conjugated anti-CD4 antibody (BD Biosciences) at 4 °C for 30 min and washed three times with PBS. These cells were fixed and permeabilized with the Cytofix/Cytoperm kit (BD Biosciences) at 4 °C for 30 min following the manufacturer’s instructions. Cells were then washed three times with Perm/Wash (BD Biosciences) and stained intracellularly with PE-conjugated anti-IFN-γ (BD Biosciences) and FITC-conjugated anti-IL-17 (BD Biosciences) at 4 °C for 30 min. After washing three times with Perm/Wash, the cells were fixed using IC fixation buffer (eBioscience, San Diego, CA, USA). Intracellular cytokine levels were detected using the software program Novocyte (Acea Biosciences, San Diego, CA, USA) and analyzed using the software program FlowJo (Treestar, Inc., San Carlos, CA, USA).
10
Intracellular Cytokine Staining for ESAT-6 Stimulation
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Lung single-cell suspensions were prepared from immunized and Mtb-infected mice and stimulated with 1 μg/mL ESAT-6 at 37°C for 12 hours in the presence of GolgiPlug and GolgiStop (BD Bioscience, San Jose, CA, USA). First, the cells were washed with PBS, and the Fc receptor was blocked with an anti-CD16/32 blocking antibody at 4 °C for 15 min. Surface molecules were stained with fluorochrome-conjugated antibodies against Thy1.2, CD4, CD44, and a Live and DEADTM Fixable Dead Cell kit for 30 min at 4 °C. After being washed with PBS, the cells were fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences, San Jose, CA, USA) for 30 min at 4 °C. The permeabilized cells were washed twice with Perm/Wash (BD Biosciences, San Jose, CA, USA) and stained with PE-conjugated anti-IFN-γ, for 30 min at 4 °C. The cells were washed twice with Perm/Wash and fixed with intracellular (IC) fixation buffer (eBioscience, San Diego, CA, USA) until analysis by flow cytometry (Beckman-Coulter, Pasadena, CA, USA).
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