Monocytes from NFAM1+/+ and NFAM1-/- mice (isolated as described above) were resuspended in cell culture media comprised of RPMI, 10% fetal bovine serum and 1% Penicillin-Streptomycin (all from Life Technologies). Monocytes were cultured in a 96 well tissue culture plate (Corning) at a concentration of 20,000 cells per well and either left untreated or pretreated for one hour with 100 µg/mL of mouse IFN-γ (R&D Systems) and subsequently stimulated with LPS (Sigma), MegaCD40L (Enzo), HKEB, HKLM, HKST, Pam3CSK4, Zymosan, FSL-1, or MDP (all from Invivogen). 24-48 hours post stimulation, supernatant was collected, and cells were harvested by flushing wells with cold PBS. Cells were analyzed by flow cytometry (see details above) and supernatants were analyzed by MSD for the presence of TNF-α (catalog #K152BHB-4), IL-6, IL-12, MIP-1α and MIP-1β (MSD custom U-plex).
Mouse ifn γ
Manufactured by R&D Systems
Sourced in United States
Mouse IFN-γ is a recombinant protein that functions as the mouse interferon-gamma cytokine. Interferon-gamma is a type II interferon that plays a key role in the immune response.
Automatically generated - may contain errors
Lab products found in correlation
Mouse il 4, by R&D Systems (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Pam3csk4, by InvivoGen (2 mentions)
Tnf α, by R&D Systems (2 mentions)
Mouse tnf α, by R&D Systems (2 mentions)
Zymosan, by InvivoGen (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
96 well tissue culture plate, by Corning (1 mentions)
Megacd40l, by Enzo Life Sciences (1 mentions)
Fsl 1, by InvivoGen (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Imidazole, by Fujifilm (1 mentions)
Rat anti mouse ifn γ mab, by R&D Systems (1 mentions)
Mouse il 2, by R&D Systems (1 mentions)
Dl glyceraldehyde, by Fujifilm (1 mentions)
β nadph, by Fujifilm (1 mentions)
Mouse il 13, by R&D Systems (1 mentions)
Gm csf, by R&D Systems (1 mentions)
21 protocols using mouse ifn γ
1
Monocyte Response to Immune Stimuli
2
Antibody-mediated Immune Cell Activation
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Special agents used in this study were as follows: recombinant human AR (Wako Chemicals USA, Inc., Richmond, VA), rabbit anti-human AR Ab (Santa Cruz Biotechnology Inc., Santa Cruz, CA), horseradish peroxidase-conjugated anti-phosphotyrosine mAb (clone RC20) (BD Transduction Laboratories, Fraklin Lakes, NJ), anti-phosphoserine mAb (Calbiochem-Novabiochem Corporation, San Diego, CA), rabbit anti-p44/42 MAP kinase Ab (Cell Signaling Technology, Inc., Danvars, MA), rabbit anti-phospho-p44/42 MAP kinase Ab (Thr202/Thr204) (Cell Signaling Technology, Inc.), rat anti-mouse CD3 mAb (Serotec Ltd., Oxford, UK), hamster anti-mouse CD28 mAb (Pharmingen Co., San Diego, CA), DynabeadsR mouse CD3/CD28 T cell expander (Invitrogen Dynal AS, Oslo, Norway), rat anti-mouse IL-2 mAb (R & D systems, Inc., minneapolis, MN), rat anti-mouse IFN-γ mAb (R & D systems), mouse IL-2 (R & D systems), mouse IFN-γ (R & D systems), D10.G4.1 T cell line (TIB 224; American Type Culture Collection (ATCC), Rockville, MD), epalrestat (Wako Pure Chemicals, Osaka, Japan), DL-glyceraldehyde (Wako), β-NADPH (Wako), imidazole (Wako), [3H]-thymidine (3H-TdR) (PerkinElmer Life Science Products Inc., Boston, MA).
3
Macrophage Polarization and Mycobacterial Infection
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Six-to eight-week-old female wild-type and TLR4-, TLR2-, and MyD88-deficient C57BL/6 J mice were used in all experiments. BMDMs were generated by flushing bone marrow cells from femurs and tibias, and culturing for 4 days in Dulbecco’s minimal essential medium (DMEM) containing 10% fetal bovine serum (FBS), penicillin (100 IU/mL), streptomycin (100 μg/mL), 25 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF; R&D Systems), or 25 ngml macrophage colony-stimulating factor (M-CSF; R&D Systems). The mouse monocyte/macrophage cell line Raw264.7 was maintained in DMEM containing 10% FBS, penicillin (100 IU/mL) and streptomycin (100 μgmL). M1 polarization of macrophages were induced by 10 ng/ml lipopolysaccharide (LPS; InvivoGen) and 10 ng/ml mouse IFN-γ (R&D Systems); M2 polarization was induced by 10 ng/ml mouse IL-4 (R&D Systems) and 10 ngml mouse IL-13 (R&D Systems). After 24 h, cells were infected with Mtb H37Rv or Mtb H37Ra (MOI=1), and incubated as described.
4
Cytokine Challenge of Osteoblasts
5
Evaluating Splenocyte Function in Tumor-Bearing Mice
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To evaluate splenocyte function, EL4 tumors borne by C57BL/6 and BALB/c-nu/nu mice were exposed to 30 Gy of X-rays. Irradiated non-tumor-bearing C57BL/6 and BALB/c-nu/nu mice were used as controls. Spleens were removed at 0, 10, 11, 14, 17, 20 and 24 days after the first tumor cell inoculation and splenocytes (3.0×106/ml) were co-cultured with irradiated EL4 cells (0.3×106/ml) in medium for 24 h. IFN-γ and TNF-α levels in the culture supernatant were then measured using mouse IFN-γ (R&D Systems, Minneapolis) and TNF-α (R&D Systems) ELISA kits, according to the manufacturers’ instructions. To measure HMGB1 concentrations, LL/C and EL4 cells were seeded in culture dishes, exposed to X-rays (Siemens-Asahi Medical Technologies, Tokyo, Japan), and then cultured in RPMI for 48 h. Culture supernatants were collected and HMGB1 concentrations were measured using an ELISA kit (Shinotest, Tokyo, Japan) according to the manufacturer’s instructions.
6
ELISA-Based Cytokine Quantification in Mouse Serum
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Mouse IFN-γ (R&D Systems) and IL-12p40 (BD Biosciences) ELISA kits were used according to the manufacturer’s protocols to determine cytokine concentrations in mouse serum. Serum concentrations of IL-4 were determined using a sandwich ELISA. Maxisorp plates (Nunc) were coated with anti-mouse IL-4 capture antibody (2 µg/mL; 11B11; BioLegend) in 50 mM sodium bicarbonate buffer (pH 9.4) overnight. Plates were washed with 0.05% Tween 20 (Sigma-Aldrich) in PBS, blocked with 4% bovine serum albumin (BSA; Roche) in PBS for 2 h, and then samples and standards (mouse rIL-4; Peprotech), serially diluted two-fold from 2,000 pg/mL, were added for 1 h. Anti-mouse IL-4 detection antibody (0.5 µg/mL; BVD6-24G2, BioLegend) diluted in 4% BSA in PBS was added for 1 h, followed by HRP-conjugated streptavidin (2 µg/mL; Sigma-Aldrich) for 30 min. The plates were developed with TMB substrate (BD Biosciences), and the reaction stopped with 1 M H2SO4 (Sigma-Aldrich).
7
Cytotoxicity Assay of Bispecific T-cell Engager
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Mouse IFN-γ (100 IU ml–1; R&D systems) was incubated with B16F10 cells overnight. Cells were labeled with 2 µM CFSE (Invitrogen) for 15 min. After washing cells with PBS, 1 µM Trp2 peptide was pulsed to 2 × 105 cells ml–1 for 1 h at 37 °C. Fivefold serial dilutions of BiTE were added to 104 cells, then mouse T cell lymphoblasts were incubated at a 1:1 E:T ratio. After incubation for 48 h, cells were resuspended in PI (ThermoFisher). CFSE and PI double-positive cell populations were gated and analyzed by Cytoflex (Beckman Coulter). For peptide titration, tenfold dilutions of Trp2 peptide and 0.5 µg ml–1 BiTE were used. Percentages of specific lysis reactions were subsequently calculated as follows: cytotoxicity (%) = (1 – (BiTE-treated units/no. of BiTE control units)) × 100.
8
Mouse IFN-γ Effects on Cell Lines
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The YUMM1.7 and YUMMER 1.7 cell lines were plated in 6-well dishes and cultured in phosphate-buffered saline (PBS, Gibco) with 1 ng/mL, 10 ng/mL, or 100 ng/mL of mouse IFN-γ (R&D Systems, Minneapolis, MN, USA) in regular culture media. All cells within the well were collected at given time-points to either be counted for total live cell numbers following trypan blue staining or stained for surface markers in subsequent flow cytometric analyses.
9
Toll-like Receptor Signaling in Inflammation
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Pam3CSK4 (TLR1/2) and LPS (TLR4) were obtained from InvivoGen (Carlsbad, CA, USA). Mouse recombinant IFN-β was purchased from PBL InterferonSource (now PBL Assay Science, Piscataway, NJ, USA) and mouse IFN-γ was from R&D Systems (Minneapolis, MN, USA). Antibodies used in this study were obtained as follows. Anti-H-PGDS and Anti-COX-2 antibodies were produced by Cayman Chemical (Ann Arbor, MI, USA). Anti-STAT1 and anti-pY701-STAT1 antibodies were from Cell Signaling Technology (Danvers, MA, USA). Anti-actin antibody was from BD Biosciences (Franklin Lakes, NJ, USA). PGs were purchased from Cayman Chemical (Ann Arbor, MI, USA). All other chemicals were from Sigma (St. Louis, MO, USA), unless stated otherwise.
10
Quantification of Serum Proteins and Cytokines
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Serum COMP (AnaMar Medical), mouse IL-18 (MBL), mouse insulin (Mercodia), mouse IFN-γ, mouse TNF-α, mouse IL-4 (all R&D systems) and (total) mouse leptin (R&D Systems) were determined using ELISA’s according to the manufacturer’s instructions. Free leptin levels were quantified by coating Maxisorp plates (Costar) overnight with mLREC purified protein (2 μg/ml in coating buffer (50 mM NaCO3; pH 10,6)). After blocking, plates were incubated with serum of treated mice. Leptin was detected with a polyclonal secondary anti-leptin Ab and streptavidin-HRP (R&D Systems).
SOL LR levels were determined as follows: a 1,000-fold dilution of mouse serum, or a serial dilution purified mLREC as a standard, was allowed to bind to penta-His Ab (Qiagen) coated Maxisorp plates (Costar). After washing, plates were incubated for 2 h at room temperature with a 1/50 dilution of a COS-1 conditioned medium containing the leptin-SEAP chimera (final concentration ±10 ng/ml). Bound secreted alkaline phosphatase activity was measured using the chemiluminescent CSPD substrate (PhosphaLight, Tropix) in a TopCount chemiluminescence counter.
SOL LR levels were determined as follows: a 1,000-fold dilution of mouse serum, or a serial dilution purified mLREC as a standard, was allowed to bind to penta-His Ab (Qiagen) coated Maxisorp plates (Costar). After washing, plates were incubated for 2 h at room temperature with a 1/50 dilution of a COS-1 conditioned medium containing the leptin-SEAP chimera (final concentration ±10 ng/ml). Bound secreted alkaline phosphatase activity was measured using the chemiluminescent CSPD substrate (PhosphaLight, Tropix) in a TopCount chemiluminescence counter.
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