Anti-mouse CD4- and CD8-coated magnetic beads were obtained from Miltenyi Biotech (Bergisch Gladbach, Germany). C57BL/6 mice were purchased from Joint Ventures Sipper BK Experimental Animal Co. (Shanghai, China) RPMI medium 1640 and FBS were purchased from PAA Laboratories (Freiburg, Germany). PE-CY7-conjugated anti-mouse CD3, APC-conjugated anti-mouse CD8, FITC-conjugated anti-mouse CD4 and PE-conjugated anti-mouse IFN-γ antibodies were purchased from eBioscience. PE-conjugated anti-mouse CD24 antibodies were purchased from Acris Antibodies (Rockville, MD, USA). Antibodies specific to p-ERK (Thr202/Tyr204), p-JNK (Thr183/Tyr185), p-p38 (Thr180/Tyr182), p-p65 (Ser536), p-PLCγ1 (Tyr783), p-Zap70 (Tyr493), p-LCK (Tyr505), LCK and β-actin were obtained from Cell Signaling Technology (Danvers, MA, USA). Trizol reagents were from Invitrogen (Carlsbad, CA, USA). Prime Script RT-PCR Kit and SYBR Premix ExTaq kit were from Takara Bio. Concanavalin A (ConA) was obtained from Sigma-Aldrich (Saint Louis, MO, USA), and α-galactosylceramide (α-GalCer) was obtained from Enzo Life Sciences (Farmingdale, NY, USA). A monoclonal antibody specific to CD4 (GK1.5) was obtained from BioXCell (West Lebanon, NH, USA).
Pe conjugated anti mouse ifn γ
Manufactured by Thermo Fisher Scientific
Sourced in United States
PE-conjugated anti-mouse IFN-γ is a laboratory reagent used to detect and quantify mouse interferon-gamma (IFN-γ) in samples. It consists of a phycoerythrin (PE) fluorescent dye conjugated to an antibody that specifically binds to mouse IFN-γ.
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Lab products found in correlation
Fitc conjugated anti mouse cd4, by Thermo Fisher Scientific (3 mentions)
α galactosylceramide α galcer, by Enzo Life Sciences (1 mentions)
Sybr premix ex taq kit, by Takara Bio (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Primescript rt pcr kit, by Takara Bio (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Murine tnf α, by Thermo Fisher Scientific (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
G csf, by Thermo Fisher Scientific (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Alum adjuvant, by Thermo Fisher Scientific (1 mentions)
Granzyme b, by Thermo Fisher Scientific (1 mentions)
Pe conjugated anti mouse il 4, by BioLegend (1 mentions)
Apc conjugated anti mouse nk1, by Thermo Fisher Scientific (1 mentions)
Recombinant mouse il 2, by Thermo Fisher Scientific (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
Pe conjugated anti mouse cd25, by Thermo Fisher Scientific (1 mentions)
4 protocols using pe conjugated anti mouse ifn γ
1
CD4+ and CD8+ T Cell Isolation
2
Murine Cytokine Expression Vectors
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Murine TNF-α, IFN-γ, G-CSF and IL-6 were purchased from PeproTech (Rocky Hill, NJ). Murine IL-2 was purchased from Millipore (Billerica, MA). PE-conjugated anti-mouse IFN-γ, PE-conjugated anti-mouse MULT-1 were purchased from eBioscience (San Diego, CA). PE-conjugated anti-mouse H60 and PE-conjugated anti-panRAE-1 were purchased from R&D Systems. Anti-asialo-GM1 mAb was purchased from Biolegend. Anti-Ly6G mAb was purchased from BioExpress (clone 1A8).
Plasmids pG, pI6, pCXCL1, and pIFN-γ are expression vectors carrying the cDNA encoding murine G-CSF, IL-6, CXCL1, and IFN-γ, respectively. Plasmid psTNF-α is the expression vector carrying the cDNA encoding IFN-β signal peptide and extracellular domain of TNF-α. These plasmids were constructed by the insertion of cDNA into plasmid pcDNA3.1 (Invitrogen, Carlsbad, CA) in our laboratory. All of the vectors were identified by in vivo expression (Ref 7 andSupplemental Figure S12 ).
Plasmids pG, pI6, pCXCL1, and pIFN-γ are expression vectors carrying the cDNA encoding murine G-CSF, IL-6, CXCL1, and IFN-γ, respectively. Plasmid psTNF-α is the expression vector carrying the cDNA encoding IFN-β signal peptide and extracellular domain of TNF-α. These plasmids were constructed by the insertion of cDNA into plasmid pcDNA3.1 (Invitrogen, Carlsbad, CA) in our laboratory. All of the vectors were identified by in vivo expression (Ref 7 and
3
Murine Th1/Th2 Immune Response Assay
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Recombinant mouse IFN-γ, anti-mouse CD3e, recombinant mouse IL-2, FITC conjugated anti-mouse CD4, PE conjugated anti-mouse IFN-γ, granzyme B, perforin, PE-Cy5 conjugated anti-mouse CD8a, APC conjugated anti-mouse NK1.1 and isotype controls of each antibody were purchased from eBioscience. FITC conjugated anti-mouse CD3e, PE conjugated anti-mouse IL-4 and IL-5 and isotype controls of each antibody were purchased from BioLegend. R-PE labeled CD1d tetramer preloaded with α-GalCer and R-PE labeled CD1d tetramer negative control were purchased from Proimmune. The mouse IFN-γ, IL-4, IL-5 and IL-13 ELISA kits were purchased from eBioscience. Alum adjuvant was purchased from Thermo Fisher Scientific. Ovalbumin was purchased from Sigma-Aldrich. The endotoxin level in Ovalbumin is < 1 EU/mg.
4
Quantifying Regulatory T Cells and Th Subsets
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To determine the number of regulatory T cells in the splenocytes, flow cytometry was performed, as previously described [7 (link)]. The splenocytes were washed with PBS and stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4 (eBioscience, San Diego, CA, USA) and phycoerythrin (PE)-conjugated anti-mouse CD25 (eBioscience, San Diego, CA, USA). Next, the cells were fixed and stained with Alexa Fluor 647 anti-mouse Foxp3 (BD Biosciences, San Jose, CA, USA) overnight at 4 °C in the dark. After washing, the cells were analyzed. In order to measure Th1 and Th17 cells in the splenocytes, FITC-conjugated anti-mouse CD4 (eBioscience, San Diego, CA, USA), PE-conjugated anti-mouse IFN-γ (eBioscience, San Diego, CA, USA), and PerCP/Cyanine5.5-conjugated anti-mouse IL-17A (eBioscience, San Diego, CA, USA) were stained according to the supplier’s instructions.
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