For Treg/Teff cell staining (Yang, et al., 2018 (link)), live/dead cells were first labeled with antibodies to distinguish living cells (Waltham, Ma, United States). The antibodies used included; anti mouse CD4 percp-cy5.5, anti-mouse cd45-bv510, anti-mouse IL-17A APC and anti-mouse IFN-γ PE, anti-mouse TNF-α Bv421, anti-mouse Foxp3 FITC (all from BioLegend). For Teff cells, cytokine staining analysis mainly containing Th17 and Th1, the cells mixed with acetate (50 ng/ml; Sigma-Aldrich), ionomycin (500 ng/ml; Sigma-Aldrich) and brefidine (1 µg/ml; Sigma-Aldrich) were cultured together and placed in 96 well plates with cytokine secretion blockers for 4 h. Stained cells were measured using BD LSR Fortessa flow cytometer (BD Biosciences) and data were obtained and were analyzed with Flowjo 10.0 (Flowjo company, United States).
Pe anti mouse ifn γ
Manufactured by BioLegend
Sourced in United States
PE anti-mouse IFN-γ is a fluorescently-labeled antibody that binds to mouse interferon-gamma (IFN-γ). It is used for the detection and quantification of mouse IFN-γ in various applications such as flow cytometry, immunohistochemistry, and ELISA.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse il 17a apc, by BioLegend (3 mentions)
Cytofix cytoperm kit, by BD (3 mentions)
Fitc anti mouse cd4, by BioLegend (3 mentions)
Fitc anti mouse cd3, by BioLegend (3 mentions)
Anti mouse cd45 bv510, by BioLegend (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Anti mouse cd4 percp cy5, by BioLegend (2 mentions)
Anti mouse tnf α bv421, by BioLegend (2 mentions)
Anti mouse foxp3 fitc, by BioLegend (2 mentions)
Apc anti mouse cd62l, by BioLegend (2 mentions)
Pe anti mouse tnf α, by BioLegend (2 mentions)
Apc anti mouse cd8a, by BioLegend (2 mentions)
Apc anti mouse cd86, by BioLegend (2 mentions)
Pe anti mouse cd86, by BioLegend (2 mentions)
Pe anti mouse cd4, by BioLegend (2 mentions)
Pe cy7 anti mouse tnf α, by BioLegend (2 mentions)
Fitc anti mouse cd8, by BioLegend (2 mentions)
Golgistop, by BD (2 mentions)
Pe anti mouse il 17a, by BioLegend (2 mentions)
Apc anti mouse cd4, by BioLegend (2 mentions)
20 protocols using pe anti mouse ifn γ
1
Treg/Teff Cell Staining and Analysis
2
Multiparametric Flow Cytometry of CD4+ T Cells
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To test the CD4+ lymphocytes, LIVE/DEAD (Thermo Fisher Scientific, Waltham, MA, USA) was first labeled to distinguish the living cells. Surface marker antimouse CD4 and CD45 were labeled to all the cultured cells. After further fixation and permeabilization were performed for intracellular staining, intracellcular inflammatory cytokines were labeled and evaluated using an LSRFortessa (BD Biosciences). The following antimouse antibodies used for the cytometry analysis were purchased from BioLegend (San Diego, CA, USA) or Abcam (Cambridge, MA, USA) including antimouse CD4 Percp-cy5.5 (Biolegend), antimouse CD45 BV510 (Biolegend), antimouse IL17A APC (Biolegend), antimouse IFN-γ PE (Biolegend), antimouse TNFα BV421 (Biolegend), antimouse Foxp3 FITC (Biolegend), antimouse pPI3K PE (Biolegend), antimouse pAKT APC (Biolegend), antimouse FoxO1 (Abcam), and antimouse pFoxO1 (Abcam). SC79 (Selleck Chemicals, Houston, TX), as an AKT activator, was also used to stimulate the cells isolated from the DLNs and spleens of the EAU group to further determine whether the PI3K/AKT/FoxO1 pathway was involved in the mechanism of apremilast treatment on EAU. All the figures and data were collected and analyzed with FlowJo software (Tree Star, Ashland, OR).
3
Urease B Protein Evaluation Assay
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Recombinant urease subunit B (rUreB) protein was purified and stored at −80°C. Peptides of 18mer and 13mer overlapping by 12 and 11 amino acids, respectively, were synthesized by GL Biochem (Shanghai, China) with purities of >90%. All peptides were dissolved in dimethyl sulfoxide (Sigma) and stored in aliquots at −80°C.
Anti-mouse CD3 (FITC), anti-mouse CD4 (APC), and anti-mouse IFN-γ (PE) antibodies were purchased from Biolegend. Anti-mouse MHC class I (H-2Kd/H-2Dd), anti-mouse MHC class II (I-A), and anti-mouse MHC class II (I-E) antibodies were purchased from eBioscience.
The adjuvants CpG OND 1826 and AddaVax were purchased from Invivogen.
Anti-mouse CD3 (FITC), anti-mouse CD4 (APC), and anti-mouse IFN-γ (PE) antibodies were purchased from Biolegend. Anti-mouse MHC class I (H-2Kd/H-2Dd), anti-mouse MHC class II (I-A), and anti-mouse MHC class II (I-E) antibodies were purchased from eBioscience.
The adjuvants CpG OND 1826 and AddaVax were purchased from Invivogen.
4
Quantitative Analysis of Virus-Specific T-Cells
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The lung and BALF cell samples were harvested at 6 days post infection from the mice immunized and challenged with A/Nanchang/H3N2 virus. The isolated lung and BALF cells were in vitro culture-stimulated with 5 µg/ml of M2e peptide and/or stalk protein with Golgi stopper (2 µg/mL). After 5 h culture, the cells were stained with mouse anti-CD3-PacificBlue (300x dilution, cat no: 100214, clone 17A2, BioLegend), anti-CD4-biotin (300x dilution, cat no: 13-0042-85, clone RM4-5, eBioscience), streptavidin-PE/Cy5 (300x dilution, cat no: 15-4317-82, eBioscience), and anti-CD8-FITC (300x dilution, cat no: MA5-16759, clone 53–6.7, eBiosciences), followed by fixation and permeabilization using a Cytofix/Cytoperm kit (10x dilution, cat no: 554715, BD Biosciences) and then, staining of intracellular cytokine using IFN-γ mAb (anti-mouse IFN-γ-PE, 200x dilution, cat no: 505808, clone XMG1.2, BioLegend). The IFN-γ+ T-cells were acquired by Becton-Dickinson LSR-II/Fortessa flow cytometer (BD) and analyzed by a sequential lymphocyte gating strategy (Supplementary Fig. S10 ) using FlowJo software (FlowJo V10, Tree Star, Inc.)45 .
5
Multiparameter Flow Cytometry Analysis
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Single-cell suspensions from mouse tissues were prepared in PBS containing 2% FBS. Cells were fixed and permeabilized with a Cytofix/Cytoperm kit (BD Biosciences) or FoxP3/Transcription Factor Staining Buffer Set (eBioscience). For surface marker analysis, cells were stained with the indicated antibodies (Abs) in PBS containing 2% FBS. Intracellular staining was performed according to the manufacturer's instructions. The following antibodies were used: anti-mouse CD4 APC-cy7, anti-mouse CD25 BV421/PE, anti-mouse FoxP3 APC, anti-mouse CD62L APC, anti-mouse CD44 PE, anti-mouse IFN-γ PE, anti-mouse IL-17A APC, anti-CD4 APC, and anti-Foxp3 PE from BioLegend. The anti-mouse/rat Ki67 was purchased from ebioscience. The anti-Akt-pSer473 rabbit antibody and anti-Akt-pThr308 rabbit antibody were purchased from Cell Signaling Technology as primary antibodies, and the donkey anti-rabbit AF594 secondary antibody was purchased from Invitrogen. Finally, the cells were washed, resuspended, and analyzed with a BD LSRFortessa X-20 flow cytometer.
6
Flow Cytometry of Immune Cell Markers
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The following reagents were utilized in the experiments: PE anti-mouse CD11c (Biolegend, Cat. No. 117308, USA), APC anti-mouse CD86 (Biolegend, Cat. No. 105008, USA), FITC anti-mouse CD4 (Biolegend, Cat. No. 100406, USA), APC anti-mouse CD8a (Biolegend, Cat. No. 100712, USA), PE anti-mouse IFN-γ (Biolegend, Cat. No. 505808, USA), and PE anti-mouse TNF-α (Biolegend, Cat. No. 506306, USA).
7
Comprehensive Immunological Profiling of Tumor Cell Lines
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4T1, H22, HepG2, NIH‐3T3, and Panc02 were purchased from BeNa Culture Collection, China. Anti‐mouse PD‐1 antibody (PD‐1 Ab, BE0146‐100 mg) was purchased from Bio X Cell. Anti‐human PD‐1 Ab was obtained as a gift from Livzon Pharmaceutical Group Inc., China. Flow cytometry antibodies included PE anti‐mouse CD274 (B7‐H1, PD‐L1) (124308), APC/Cyanine7 anti‐mouse CD45 (103132), FITC anti‐mouse CD3 (100204), Brilliant Violet 421 anti‐mouse CD4 (100563), PE/Cy7 anti‐mouse CD8a (100722), PE anti‐mouse IFN‐γ (505808), Alexa Fluor 647 anti‐human/mouse Granzyme B (515406), APC anti‐mouse Ki‐67 (652406), PE anti‐mouse/human CD44 (103008), PE anti‐mouse Ly‐6G/Ly‐6C (Gr‐1) (108407), APC/Cyanine7 anti‐mouse/human CD11b (101226), Brilliant Violet 421 anti‐mouse F4/80 (123137), PE anti‐mouse CD86 (105008), PE/Cy7 anti‐mouse CD206 (MMR) (141720), Alexa Fluor 647 anti‐mouse FOXP3 (126408), APC anti‐mouse CD49b (pan‐NK cells) (108910), PE anti‐mouse CD25 (102008), PE anti‐mouse/human CD44 (103008), APC anti‐mouse CD62L (104412), Zombie Aqua Fixable Viability Kit (423102), Cell Activation Cocktail (with Brefeldin A) (423304), True‐Nuclear Transcription Factor Buffer Set (424401), and Intracellular Staining Permeabilization Wash Buffer (10X) (421002) were purchased from BioLegend.
8
Single Cell Immune Profiling Protocol
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Single cell suspensions from the spleen and liver were acquired according to the methods previously described (21 (link)) and analyzed using flow cytometry. Antibodies used for flow cytometry staining including Percp-Cy5.5-anti-mouse-CD45.1, APC-Cy7-anti-mouse-CD11b, Percp-Cy5.5-anti-mouse-NK1.1, BV650-anti-mouse-H2-Kb were purchased from BD Bioscience (SanDiego, CA, USA); purified anti-mouse-CD16/32, APC-anti-mouseCD43, PE-anti-mouse-NKp46, APC-anti-mouse-CD107, FITC-anti-mouse-NKG2D, PE-anti-mouse-IFN-γ, PE/Cy7-anti-mouse-TNF-α, FITC-anti-mouse-CD62E, PE-anti-mouse-CD4, PE/Cy7-anti-mouse-CD44, APC/Cy7-anti-mouse-CD62L, Pacific Blue-anti-mouse-CD8a, FITC-anti-mouse-CD69 were purchased from Biolegend (San Diego, CA, USA); PE-Cy7-anti-mouse-Granzyme B, APC-anti-mouse-Perforin were purchased from eBioscience (San Diego, CA, USA). Recombinant Mouse E-Selectin, P-Selectin, and L-Selectin chimera were purchased from Biolegend (San Diego, CA, USA) for detecting the binding abilities. Samples were detected on a NovoCyte Flow Cytometer (ACEA Biosciences, San Diego, CA, USA) and data were analyzed by using Flowjo software (Flowjo, Ashland, OR, USA).
9
Evaluating IFN-γ Response to Cas9 Delivery
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Mice were injected thrice with empty LNPs, sgRNA/Cas9 mRNA–encapsulated LNPs, and PBS. Twenty-four hours after the last injection, single-cell suspensions were prepared from mice spleens. Cells (1 × 106) were cultured with medium alone (negative control), 2.5 μg of recombinant Cas9 (Cas9), or phorbol 12-myristate 13-acetate (PMA) (50 ng/ml) and ionomycin (1 μg/ml) (positive control) for 18 hours. To measure the IFN-γ–producing cells, splenocytes were further treated with brefeldin A (eBioscience Inc., San Diego, CA, USA) for 5 hours. Cells were then permeabilized with fluorescence-activated cell sorting (FACS) buffer containing 0.5% saponin (Sigma-Aldrich, St. Louis, MO, USA) and stained with markers. The stained cells were acquired using a CytoFLEX S flow cytometer (Beckman Coulter, Brea, CA, USA) and analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA). The antibodies used were antigen-presenting cell (APC)/Cy7 anti-mouse CD3 (BioLegend, San Diego, CA, USA, 100706), fluorescein isothiocyanate (FITC) anti-mouse CD8 (BioLegend, 100222), and phycoerythrin (PE) anti-mouse IFN-γ (BioLegend, 505808).
10
Intracellular Cytokine Analysis Protocol
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Cytokine production was assessed with BD Cytofix/Cytoperm containing BD Golgi-Plug (BD Biosciences). Cells were stimulated with phorbol 12-myristate 13-acetate (PMA, 50 ng/ml, Sigma), Ionomycin (1 μg/ml, Sigma), and GolgiStop (1 μl/ml, BD Biosciences) at 37°C in 5% CO2 for 4 h. After surface staining, cells were fixed, permeabilized, and stained for IFN-γ (PE-anti-mouse IFN-γ, Biolegend), IL-4 (PE-anti-mouse IL-4, Biolegend), and IL-17 (PE-anti-mouse IL-17A, Biolegend). For intracellular staining IL-21, permeabilized cells were incubated with IL-21R/Fc chimera (R&D systems) for 1 h at 4°C. Cells were then washed and stained with PE-conjugated affinity-purified F(ab')2 fragment of goat anti-human Fc γ antibody (Jackson ImmunoResearch Laboratories) for 30 min at 4°C. Viability was assessed using LIVE/DEAD Cell Viability Assays (Life Technologies).
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