Recombinant:Article Title: Novel roles of dense granule protein 12 (GRA12) in Toxoplasma gondii infection
Article Snippet: .. Mouse macrophage RAW264.7 cells were activated by treatment with 0.1 ng/mL LPS (Escherichia coli 055:B5; SigmaAldrich) and 100 U/mL murine recombinant IFN-γ (rIFN-γ; R&D Systems) for 8 hours, as previously described.20 Activated cells were infected with freshly harvested tachyzoites for 2 hours, after which cultures were fixed and stained | 5WANG et Al. using mouse anti-GRA5 for staining the PV and rabbit antiIrga6 for immunity-related GTPase localization. ..
Article Title: Tbc1d10c is a selective, constitutive suppressor of the CD8 T-cell anti-tumor response
Article Snippet: SCC1 and LungMet2 cells were cultured in complete FAD medium.42 MC38, B16, and B16-OVA cells were cultured in DMEM (Gibco) supplemented with 10% defined FBS (HyClone). .. In some cases, B16-OVA cells were incubated with 10 ng/mL murine recombinant IFN-γ (R&D Systems) for 24 hours to stimulate MHC I expression.43 Murine and human T cells were cultured in RPMI 1640-GlutaMax (Gibco) supplemented with 10% heat-inactivated defined FBS (HyClone) and 55 μM 2-Mercaptoethanol (Gibco). siRNA Gene Silencing Primary murine or human CD8 T cells were electroporated with four pooled (SMARTpool) pre-designed siRNAs (Dharmacon) targeting murine Epsti1, Map3k3 or human TBC1D10C or non-targeting siRNA control (Scramble) using Mouse or Human T Cell Nucleofector Kits as per manufacturer’s instruction (Lonza). ..
Article Title: Endogenous SLPI contributes to the regulation of inflammatory responses in peritoneal macrophages by modulating MMP-9 production
Article Snippet: .. The following reagents were used: Alhydrogel (Cat. No. vac-alu-250) referred as “alum”, ultrapure LPS from E. coli serotype O55:B5 (Cat. No. Tlrl-pb5lps) for in vivo experiments, mouse TLR1–9 Agonist Kit (Cat. No. tlrl-kit1mw), nigericin (Cat. No. tlrl-nig) all from Invivogen (Toulouse, France); BD DIFCO fluid thioglycolate medium (Cat. No. 225650; BD, Argenta, Poznan, Poland); TMB substrate reagent set (Cat. No. 555214; BD, Warsaw, Poland), streptavidin PE (Cat. No. 554061; BD, Warsaw, Poland); LPS from E. coli serotype EH100 (Cat. No. ALX-581-010-L001; Enzo, Farmingdale, USA) for in vitro experiments; murine recombinant IFN-γ (Cat. No. 485-MI; R&D Systems, Bio-Techne, Warsaw, Poland); Peprotech murine recombinant IL-4 (Cat. No. 214-14), Peprotech murine recombinant M-CSF (Cat. No. 315-02), Gibco DMEM with high glucose (Cat. No. 11965092), penicillin-streptomycin (Cat. No. 15070063), Gibco heat-inactivated fetal bovine serum (Cat. No. 10500064), eBioscience Intracellular Fixation Buffer (Cat. No. 00-8222-49), eBioscience Foxp3/Transcription Factor Fixation/Permeabilization concentrate and diluent (Cat. No. 00-5521-00), eBioscience permeabilization buffer (10X) (Cat. No. 00-8333-56) all from Thermo Fisher Scientific (Life Technologies, Warsaw, Poland); inhibitors: JNK-IN-8 (Cat. No. HY-13319), SP600125 (Cat. No. HY-12041), PD98059 (Cat. No. HY-12028) and SB203580 (Cat. No. HY-10256) all from MedChemExpress (Sollentuna, Sweden); PBS (Cat. No. P04-36500; PAN-Biotech, Immuniq, Zory, Poland); sodium chloride injection solution (Cat. No. 3117301, Polpharma, Starogard Gdanski, Poland); BSA (Cat. No. ALB004; BioShop, Epro Science, Wladyslawowo, Poland); Tween-20 (Cat. No. P9416), saponin (Cat. No. 84510), gelatin type A from porcine skin (Cat. No. G-2625) all from Sigma-Aldrich (Merck Life Science, Poznan, Poland); Triton X-100 (Cat. No. 841810492; Poch SA, Gliwice, Poland); 2x Laemmli sample buffer (Cat. No. 1610737; Bio-Rad, Warsaw, Poland); WesternBright ECL HRP substrate (Cat. No. K-12045-D20, Advansta, San Jose, California, USA); paraformaldehyde (Cat. No. 43368.9M; VWR International, Gdansk, Poland); Zombie Aqua Fixable Viability Kit (Cat. No. 423102; Biolegend, Amsterdam, The Netherlands). .. The following antibodies were used: anti-CD3 PerCPCy5.5 (0.1 μg/100 μl; clone 17A2, Cat. No. 100218), anti-F4/80 Alexa Fluor 647 (0.25 μg/100 μl; clone BM8, Cat. No. 123122), anti-CD45 APCCy7 (0.2 μg/100 μl; clone 104; Cat. No. 109824), anti-Ly6C PE-Cy7 (0.005 μg/ml; clone HK1.4, Cat. No. 128017), anti-Ly6G FITC (0.25 μg/100 μl; clone 1A8, Cat. No. 127605) all from Biolegend (Amsterdam, The Netherlands); anti-CD11b BV650 (0.04 μg/100 μl; clone M1/70; Cat. No. 563402), anti-CD11c BV421 (0.2 μg/100 μl; clone HL3; Cat. No. 562782), anti-Ly6G BV711 (0.2 μg/100 μl; clone 1A8; Cat. No. 563979), anti-SiglecF PerCPCy5.5 (0.1 μg/100 μl; clone E50-2440; Cat. No. 565526) all from BD (Warsaw, Poland); anti-CD16/CD32 monoclonal antibodies (0.25 μg/100 μl; clone 93; Cat. No. 14-0161-85), eBioscience anti-c-kit FITC (0.25 μg/100 μl; clone 2B8; Cat. No. 11-1171-85), anti-CD11c PerCPCy5.5 (0.1 μg/100 μl; clone N418, Cat. No. 45-0114), anti-CD19 PerCPCy5.5 (0.1 μg/100 μl; clone 1D3, Cat. No. 45-0193-82), anti-MHC II Alexa Fluor 700 (0.02 μg/100 μl; clone M5/114.15.2, Cat. No. 56-5321-80), anti-SiglecF eFluor660 (0.04 μg/100 μl; clone 1RNM44N, Cat. No. 50-1702-82) all from Thermo Fisher Scientific (Life Technologies, Warsaw, Poland); anti-mouse SLPI – biotin (0.067 μg/100μl; Cat. No. BAF-1735), anti-mouse SLPI (2 μg/ml; Cat. No. AF1735), anti-mouse MMP9 (0.25 μg/ml; Cat. No. AF909-SP) all from R&D Systems (Bio-Techne, Warsaw, Poland); anti-β-actin (1:5000 dilution; clone AC15; Cat. No. A1978), rabbit anti-goat IgG antibody HRP conjugated (0.5 μg/ml; Cat. No. AP106P) all from Sigma-Aldrich (Merck Life Science, Poznan, Poland); StarBright Blue 520 goat anti-mouse IgG (1:2500 dilution; Cat. No. 12005867, Bio-Rad, Warsaw, Poland).
Article Title: Tbc1d10c is a selective, constitutive suppressor of the CD8 T-cell anti-tumor response
Article Snippet: .. MC38 cells were pre-incubated with 10 ng/ml murine recombinant Ifn-γ (R&D Systems) and OVA257-264 peptide for 24 hrs and subsequently stained with PKH26 membrane dye as per manufacturer’s protocol (Sigma Aldrich). ..
Article Title: Soluble epoxide hydrolase promotes astrocyte survival in retinopathy of prematurity
Article Snippet: Using a Dynal magnetic tube holder, cells not bound to magnetic beads were collected and washed in DMEM containing 10% FBS. .. Cells were plated in fibronectin-coated (2 μg/mL in serum-free DMEM; BD Biosciences) dishes and cultured in DMEM containing 10% FBS, 2-mM L-glutamine, 2-mM sodium pyruvate, 20-mM HEPES, 1% nonessential amino acids, 100 μg/mL streptomycin, 100 U/mL penicillin, 55 U/mL heparin (Sigma-Aldrich), endothelial growth supplement 100 μg/mL (Sigma-Aldrich), and the murine recombinant IFN-γ (R&D Systems) at 44 U/mL and incubated at 37°C with 5% CO 2 . ..
Article Title: Tbc1d10c is a selective, constitutive suppressor of the CD8 T-cell anti-tumor response
Article Snippet: MC38, B16, and B16-OVA cells were cultured in DMEM (Gibco) supplemented with 10% defined FBS (HyClone). .. In some cases, B16-OVA cells were incubated with 10 ng/mL murine recombinant IFN-γ (R&D Systems) for 24 hours to stimulate MHC I expression. .. Murine and human T cells were cultured in RPMI 1640-GlutaMax (Gibco) supplemented with 10% heat-inactivated defined FBS (HyClone) and 55 μM 2-Mercaptoethanol (Gibco).
Infection:Article Title: Novel roles of dense granule protein 12 (GRA12) in Toxoplasma gondii infection
Article Snippet: .. Mouse macrophage RAW264.7 cells were activated by treatment with 0.1 ng/mL LPS (Escherichia coli 055:B5; SigmaAldrich) and 100 U/mL murine recombinant IFN-γ (rIFN-γ; R&D Systems) for 8 hours, as previously described.20 Activated cells were infected with freshly harvested tachyzoites for 2 hours, after which cultures were fixed and stained | 5WANG et Al. using mouse anti-GRA5 for staining the PV and rabbit antiIrga6 for immunity-related GTPase localization. ..
Staining:Article Title: Novel roles of dense granule protein 12 (GRA12) in Toxoplasma gondii infection
Article Snippet: .. Mouse macrophage RAW264.7 cells were activated by treatment with 0.1 ng/mL LPS (Escherichia coli 055:B5; SigmaAldrich) and 100 U/mL murine recombinant IFN-γ (rIFN-γ; R&D Systems) for 8 hours, as previously described.20 Activated cells were infected with freshly harvested tachyzoites for 2 hours, after which cultures were fixed and stained | 5WANG et Al. using mouse anti-GRA5 for staining the PV and rabbit antiIrga6 for immunity-related GTPase localization. ..
Article Title: Tbc1d10c is a selective, constitutive suppressor of the CD8 T-cell anti-tumor response
Article Snippet: .. MC38 cells were pre-incubated with 10 ng/ml murine recombinant Ifn-γ (R&D Systems) and OVA257-264 peptide for 24 hrs and subsequently stained with PKH26 membrane dye as per manufacturer’s protocol (Sigma Aldrich). ..
Incubation:Article Title: Tbc1d10c is a selective, constitutive suppressor of the CD8 T-cell anti-tumor response
Article Snippet: SCC1 and LungMet2 cells were cultured in complete FAD medium.42 MC38, B16, and B16-OVA cells were cultured in DMEM (Gibco) supplemented with 10% defined FBS (HyClone). .. In some cases, B16-OVA cells were incubated with 10 ng/mL murine recombinant IFN-γ (R&D Systems) for 24 hours to stimulate MHC I expression.43 Murine and human T cells were cultured in RPMI 1640-GlutaMax (Gibco) supplemented with 10% heat-inactivated defined FBS (HyClone) and 55 μM 2-Mercaptoethanol (Gibco). siRNA Gene Silencing Primary murine or human CD8 T cells were electroporated with four pooled (SMARTpool) pre-designed siRNAs (Dharmacon) targeting murine Epsti1, Map3k3 or human TBC1D10C or non-targeting siRNA control (Scramble) using Mouse or Human T Cell Nucleofector Kits as per manufacturer’s instruction (Lonza). ..
Article Title: Soluble epoxide hydrolase promotes astrocyte survival in retinopathy of prematurity
Article Snippet: Using a Dynal magnetic tube holder, cells not bound to magnetic beads were collected and washed in DMEM containing 10% FBS. .. Cells were plated in fibronectin-coated (2 μg/mL in serum-free DMEM; BD Biosciences) dishes and cultured in DMEM containing 10% FBS, 2-mM L-glutamine, 2-mM sodium pyruvate, 20-mM HEPES, 1% nonessential amino acids, 100 μg/mL streptomycin, 100 U/mL penicillin, 55 U/mL heparin (Sigma-Aldrich), endothelial growth supplement 100 μg/mL (Sigma-Aldrich), and the murine recombinant IFN-γ (R&D Systems) at 44 U/mL and incubated at 37°C with 5% CO 2 . ..
Article Title: Tbc1d10c is a selective, constitutive suppressor of the CD8 T-cell anti-tumor response
Article Snippet: MC38, B16, and B16-OVA cells were cultured in DMEM (Gibco) supplemented with 10% defined FBS (HyClone). .. In some cases, B16-OVA cells were incubated with 10 ng/mL murine recombinant IFN-γ (R&D Systems) for 24 hours to stimulate MHC I expression. .. Murine and human T cells were cultured in RPMI 1640-GlutaMax (Gibco) supplemented with 10% heat-inactivated defined FBS (HyClone) and 55 μM 2-Mercaptoethanol (Gibco).
Immunopeptidomics:Article Title: Tbc1d10c is a selective, constitutive suppressor of the CD8 T-cell anti-tumor response
Article Snippet: SCC1 and LungMet2 cells were cultured in complete FAD medium.42 MC38, B16, and B16-OVA cells were cultured in DMEM (Gibco) supplemented with 10% defined FBS (HyClone). .. In some cases, B16-OVA cells were incubated with 10 ng/mL murine recombinant IFN-γ (R&D Systems) for 24 hours to stimulate MHC I expression.43 Murine and human T cells were cultured in RPMI 1640-GlutaMax (Gibco) supplemented with 10% heat-inactivated defined FBS (HyClone) and 55 μM 2-Mercaptoethanol (Gibco). siRNA Gene Silencing Primary murine or human CD8 T cells were electroporated with four pooled (SMARTpool) pre-designed siRNAs (Dharmacon) targeting murine Epsti1, Map3k3 or human TBC1D10C or non-targeting siRNA control (Scramble) using Mouse or Human T Cell Nucleofector Kits as per manufacturer’s instruction (Lonza). ..
Article Title: Tbc1d10c is a selective, constitutive suppressor of the CD8 T-cell anti-tumor response
Article Snippet: MC38, B16, and B16-OVA cells were cultured in DMEM (Gibco) supplemented with 10% defined FBS (HyClone). .. In some cases, B16-OVA cells were incubated with 10 ng/mL murine recombinant IFN-γ (R&D Systems) for 24 hours to stimulate MHC I expression. .. Murine and human T cells were cultured in RPMI 1640-GlutaMax (Gibco) supplemented with 10% heat-inactivated defined FBS (HyClone) and 55 μM 2-Mercaptoethanol (Gibco).
Expressing:Article Title: Tbc1d10c is a selective, constitutive suppressor of the CD8 T-cell anti-tumor response
Article Snippet: SCC1 and LungMet2 cells were cultured in complete FAD medium.42 MC38, B16, and B16-OVA cells were cultured in DMEM (Gibco) supplemented with 10% defined FBS (HyClone). .. In some cases, B16-OVA cells were incubated with 10 ng/mL murine recombinant IFN-γ (R&D Systems) for 24 hours to stimulate MHC I expression.43 Murine and human T cells were cultured in RPMI 1640-GlutaMax (Gibco) supplemented with 10% heat-inactivated defined FBS (HyClone) and 55 μM 2-Mercaptoethanol (Gibco). siRNA Gene Silencing Primary murine or human CD8 T cells were electroporated with four pooled (SMARTpool) pre-designed siRNAs (Dharmacon) targeting murine Epsti1, Map3k3 or human TBC1D10C or non-targeting siRNA control (Scramble) using Mouse or Human T Cell Nucleofector Kits as per manufacturer’s instruction (Lonza). ..
Article Title: Tbc1d10c is a selective, constitutive suppressor of the CD8 T-cell anti-tumor response
Article Snippet: MC38, B16, and B16-OVA cells were cultured in DMEM (Gibco) supplemented with 10% defined FBS (HyClone). .. In some cases, B16-OVA cells were incubated with 10 ng/mL murine recombinant IFN-γ (R&D Systems) for 24 hours to stimulate MHC I expression. .. Murine and human T cells were cultured in RPMI 1640-GlutaMax (Gibco) supplemented with 10% heat-inactivated defined FBS (HyClone) and 55 μM 2-Mercaptoethanol (Gibco).
Cell Culture:Article Title: Tbc1d10c is a selective, constitutive suppressor of the CD8 T-cell anti-tumor response
Article Snippet: SCC1 and LungMet2 cells were cultured in complete FAD medium.42 MC38, B16, and B16-OVA cells were cultured in DMEM (Gibco) supplemented with 10% defined FBS (HyClone). .. In some cases, B16-OVA cells were incubated with 10 ng/mL murine recombinant IFN-γ (R&D Systems) for 24 hours to stimulate MHC I expression.43 Murine and human T cells were cultured in RPMI 1640-GlutaMax (Gibco) supplemented with 10% heat-inactivated defined FBS (HyClone) and 55 μM 2-Mercaptoethanol (Gibco). siRNA Gene Silencing Primary murine or human CD8 T cells were electroporated with four pooled (SMARTpool) pre-designed siRNAs (Dharmacon) targeting murine Epsti1, Map3k3 or human TBC1D10C or non-targeting siRNA control (Scramble) using Mouse or Human T Cell Nucleofector Kits as per manufacturer’s instruction (Lonza). ..
Article Title: Soluble epoxide hydrolase promotes astrocyte survival in retinopathy of prematurity
Article Snippet: Using a Dynal magnetic tube holder, cells not bound to magnetic beads were collected and washed in DMEM containing 10% FBS. .. Cells were plated in fibronectin-coated (2 μg/mL in serum-free DMEM; BD Biosciences) dishes and cultured in DMEM containing 10% FBS, 2-mM L-glutamine, 2-mM sodium pyruvate, 20-mM HEPES, 1% nonessential amino acids, 100 μg/mL streptomycin, 100 U/mL penicillin, 55 U/mL heparin (Sigma-Aldrich), endothelial growth supplement 100 μg/mL (Sigma-Aldrich), and the murine recombinant IFN-γ (R&D Systems) at 44 U/mL and incubated at 37°C with 5% CO 2 . ..
Control:Article Title: Tbc1d10c is a selective, constitutive suppressor of the CD8 T-cell anti-tumor response
Article Snippet: SCC1 and LungMet2 cells were cultured in complete FAD medium.42 MC38, B16, and B16-OVA cells were cultured in DMEM (Gibco) supplemented with 10% defined FBS (HyClone). .. In some cases, B16-OVA cells were incubated with 10 ng/mL murine recombinant IFN-γ (R&D Systems) for 24 hours to stimulate MHC I expression.43 Murine and human T cells were cultured in RPMI 1640-GlutaMax (Gibco) supplemented with 10% heat-inactivated defined FBS (HyClone) and 55 μM 2-Mercaptoethanol (Gibco). siRNA Gene Silencing Primary murine or human CD8 T cells were electroporated with four pooled (SMARTpool) pre-designed siRNAs (Dharmacon) targeting murine Epsti1, Map3k3 or human TBC1D10C or non-targeting siRNA control (Scramble) using Mouse or Human T Cell Nucleofector Kits as per manufacturer’s instruction (Lonza). ..
In Vivo:Article Title: Endogenous SLPI contributes to the regulation of inflammatory responses in peritoneal macrophages by modulating MMP-9 production
Article Snippet: .. The following reagents were used: Alhydrogel (Cat. No. vac-alu-250) referred as “alum”, ultrapure LPS from E. coli serotype O55:B5 (Cat. No. Tlrl-pb5lps) for in vivo experiments, mouse TLR1–9 Agonist Kit (Cat. No. tlrl-kit1mw), nigericin (Cat. No. tlrl-nig) all from Invivogen (Toulouse, France); BD DIFCO fluid thioglycolate medium (Cat. No. 225650; BD, Argenta, Poznan, Poland); TMB substrate reagent set (Cat. No. 555214; BD, Warsaw, Poland), streptavidin PE (Cat. No. 554061; BD, Warsaw, Poland); LPS from E. coli serotype EH100 (Cat. No. ALX-581-010-L001; Enzo, Farmingdale, USA) for in vitro experiments; murine recombinant IFN-γ (Cat. No. 485-MI; R&D Systems, Bio-Techne, Warsaw, Poland); Peprotech murine recombinant IL-4 (Cat. No. 214-14), Peprotech murine recombinant M-CSF (Cat. No. 315-02), Gibco DMEM with high glucose (Cat. No. 11965092), penicillin-streptomycin (Cat. No. 15070063), Gibco heat-inactivated fetal bovine serum (Cat. No. 10500064), eBioscience Intracellular Fixation Buffer (Cat. No. 00-8222-49), eBioscience Foxp3/Transcription Factor Fixation/Permeabilization concentrate and diluent (Cat. No. 00-5521-00), eBioscience permeabilization buffer (10X) (Cat. No. 00-8333-56) all from Thermo Fisher Scientific (Life Technologies, Warsaw, Poland); inhibitors: JNK-IN-8 (Cat. No. HY-13319), SP600125 (Cat. No. HY-12041), PD98059 (Cat. No. HY-12028) and SB203580 (Cat. No. HY-10256) all from MedChemExpress (Sollentuna, Sweden); PBS (Cat. No. P04-36500; PAN-Biotech, Immuniq, Zory, Poland); sodium chloride injection solution (Cat. No. 3117301, Polpharma, Starogard Gdanski, Poland); BSA (Cat. No. ALB004; BioShop, Epro Science, Wladyslawowo, Poland); Tween-20 (Cat. No. P9416), saponin (Cat. No. 84510), gelatin type A from porcine skin (Cat. No. G-2625) all from Sigma-Aldrich (Merck Life Science, Poznan, Poland); Triton X-100 (Cat. No. 841810492; Poch SA, Gliwice, Poland); 2x Laemmli sample buffer (Cat. No. 1610737; Bio-Rad, Warsaw, Poland); WesternBright ECL HRP substrate (Cat. No. K-12045-D20, Advansta, San Jose, California, USA); paraformaldehyde (Cat. No. 43368.9M; VWR International, Gdansk, Poland); Zombie Aqua Fixable Viability Kit (Cat. No. 423102; Biolegend, Amsterdam, The Netherlands). .. The following antibodies were used: anti-CD3 PerCPCy5.5 (0.1 μg/100 μl; clone 17A2, Cat. No. 100218), anti-F4/80 Alexa Fluor 647 (0.25 μg/100 μl; clone BM8, Cat. No. 123122), anti-CD45 APCCy7 (0.2 μg/100 μl; clone 104; Cat. No. 109824), anti-Ly6C PE-Cy7 (0.005 μg/ml; clone HK1.4, Cat. No. 128017), anti-Ly6G FITC (0.25 μg/100 μl; clone 1A8, Cat. No. 127605) all from Biolegend (Amsterdam, The Netherlands); anti-CD11b BV650 (0.04 μg/100 μl; clone M1/70; Cat. No. 563402), anti-CD11c BV421 (0.2 μg/100 μl; clone HL3; Cat. No. 562782), anti-Ly6G BV711 (0.2 μg/100 μl; clone 1A8; Cat. No. 563979), anti-SiglecF PerCPCy5.5 (0.1 μg/100 μl; clone E50-2440; Cat. No. 565526) all from BD (Warsaw, Poland); anti-CD16/CD32 monoclonal antibodies (0.25 μg/100 μl; clone 93; Cat. No. 14-0161-85), eBioscience anti-c-kit FITC (0.25 μg/100 μl; clone 2B8; Cat. No. 11-1171-85), anti-CD11c PerCPCy5.5 (0.1 μg/100 μl; clone N418, Cat. No. 45-0114), anti-CD19 PerCPCy5.5 (0.1 μg/100 μl; clone 1D3, Cat. No. 45-0193-82), anti-MHC II Alexa Fluor 700 (0.02 μg/100 μl; clone M5/114.15.2, Cat. No. 56-5321-80), anti-SiglecF eFluor660 (0.04 μg/100 μl; clone 1RNM44N, Cat. No. 50-1702-82) all from Thermo Fisher Scientific (Life Technologies, Warsaw, Poland); anti-mouse SLPI – biotin (0.067 μg/100μl; Cat. No. BAF-1735), anti-mouse SLPI (2 μg/ml; Cat. No. AF1735), anti-mouse MMP9 (0.25 μg/ml; Cat. No. AF909-SP) all from R&D Systems (Bio-Techne, Warsaw, Poland); anti-β-actin (1:5000 dilution; clone AC15; Cat. No. A1978), rabbit anti-goat IgG antibody HRP conjugated (0.5 μg/ml; Cat. No. AP106P) all from Sigma-Aldrich (Merck Life Science, Poznan, Poland); StarBright Blue 520 goat anti-mouse IgG (1:2500 dilution; Cat. No. 12005867, Bio-Rad, Warsaw, Poland).
In Vitro:Article Title: Endogenous SLPI contributes to the regulation of inflammatory responses in peritoneal macrophages by modulating MMP-9 production
Article Snippet: .. The following reagents were used: Alhydrogel (Cat. No. vac-alu-250) referred as “alum”, ultrapure LPS from E. coli serotype O55:B5 (Cat. No. Tlrl-pb5lps) for in vivo experiments, mouse TLR1–9 Agonist Kit (Cat. No. tlrl-kit1mw), nigericin (Cat. No. tlrl-nig) all from Invivogen (Toulouse, France); BD DIFCO fluid thioglycolate medium (Cat. No. 225650; BD, Argenta, Poznan, Poland); TMB substrate reagent set (Cat. No. 555214; BD, Warsaw, Poland), streptavidin PE (Cat. No. 554061; BD, Warsaw, Poland); LPS from E. coli serotype EH100 (Cat. No. ALX-581-010-L001; Enzo, Farmingdale, USA) for in vitro experiments; murine recombinant IFN-γ (Cat. No. 485-MI; R&D Systems, Bio-Techne, Warsaw, Poland); Peprotech murine recombinant IL-4 (Cat. No. 214-14), Peprotech murine recombinant M-CSF (Cat. No. 315-02), Gibco DMEM with high glucose (Cat. No. 11965092), penicillin-streptomycin (Cat. No. 15070063), Gibco heat-inactivated fetal bovine serum (Cat. No. 10500064), eBioscience Intracellular Fixation Buffer (Cat. No. 00-8222-49), eBioscience Foxp3/Transcription Factor Fixation/Permeabilization concentrate and diluent (Cat. No. 00-5521-00), eBioscience permeabilization buffer (10X) (Cat. No. 00-8333-56) all from Thermo Fisher Scientific (Life Technologies, Warsaw, Poland); inhibitors: JNK-IN-8 (Cat. No. HY-13319), SP600125 (Cat. No. HY-12041), PD98059 (Cat. No. HY-12028) and SB203580 (Cat. No. HY-10256) all from MedChemExpress (Sollentuna, Sweden); PBS (Cat. No. P04-36500; PAN-Biotech, Immuniq, Zory, Poland); sodium chloride injection solution (Cat. No. 3117301, Polpharma, Starogard Gdanski, Poland); BSA (Cat. No. ALB004; BioShop, Epro Science, Wladyslawowo, Poland); Tween-20 (Cat. No. P9416), saponin (Cat. No. 84510), gelatin type A from porcine skin (Cat. No. G-2625) all from Sigma-Aldrich (Merck Life Science, Poznan, Poland); Triton X-100 (Cat. No. 841810492; Poch SA, Gliwice, Poland); 2x Laemmli sample buffer (Cat. No. 1610737; Bio-Rad, Warsaw, Poland); WesternBright ECL HRP substrate (Cat. No. K-12045-D20, Advansta, San Jose, California, USA); paraformaldehyde (Cat. No. 43368.9M; VWR International, Gdansk, Poland); Zombie Aqua Fixable Viability Kit (Cat. No. 423102; Biolegend, Amsterdam, The Netherlands). .. The following antibodies were used: anti-CD3 PerCPCy5.5 (0.1 μg/100 μl; clone 17A2, Cat. No. 100218), anti-F4/80 Alexa Fluor 647 (0.25 μg/100 μl; clone BM8, Cat. No. 123122), anti-CD45 APCCy7 (0.2 μg/100 μl; clone 104; Cat. No. 109824), anti-Ly6C PE-Cy7 (0.005 μg/ml; clone HK1.4, Cat. No. 128017), anti-Ly6G FITC (0.25 μg/100 μl; clone 1A8, Cat. No. 127605) all from Biolegend (Amsterdam, The Netherlands); anti-CD11b BV650 (0.04 μg/100 μl; clone M1/70; Cat. No. 563402), anti-CD11c BV421 (0.2 μg/100 μl; clone HL3; Cat. No. 562782), anti-Ly6G BV711 (0.2 μg/100 μl; clone 1A8; Cat. No. 563979), anti-SiglecF PerCPCy5.5 (0.1 μg/100 μl; clone E50-2440; Cat. No. 565526) all from BD (Warsaw, Poland); anti-CD16/CD32 monoclonal antibodies (0.25 μg/100 μl; clone 93; Cat. No. 14-0161-85), eBioscience anti-c-kit FITC (0.25 μg/100 μl; clone 2B8; Cat. No. 11-1171-85), anti-CD11c PerCPCy5.5 (0.1 μg/100 μl; clone N418, Cat. No. 45-0114), anti-CD19 PerCPCy5.5 (0.1 μg/100 μl; clone 1D3, Cat. No. 45-0193-82), anti-MHC II Alexa Fluor 700 (0.02 μg/100 μl; clone M5/114.15.2, Cat. No. 56-5321-80), anti-SiglecF eFluor660 (0.04 μg/100 μl; clone 1RNM44N, Cat. No. 50-1702-82) all from Thermo Fisher Scientific (Life Technologies, Warsaw, Poland); anti-mouse SLPI – biotin (0.067 μg/100μl; Cat. No. BAF-1735), anti-mouse SLPI (2 μg/ml; Cat. No. AF1735), anti-mouse MMP9 (0.25 μg/ml; Cat. No. AF909-SP) all from R&D Systems (Bio-Techne, Warsaw, Poland); anti-β-actin (1:5000 dilution; clone AC15; Cat. No. A1978), rabbit anti-goat IgG antibody HRP conjugated (0.5 μg/ml; Cat. No. AP106P) all from Sigma-Aldrich (Merck Life Science, Poznan, Poland); StarBright Blue 520 goat anti-mouse IgG (1:2500 dilution; Cat. No. 12005867, Bio-Rad, Warsaw, Poland).
Injection:Article Title: Endogenous SLPI contributes to the regulation of inflammatory responses in peritoneal macrophages by modulating MMP-9 production
Article Snippet: .. The following reagents were used: Alhydrogel (Cat. No. vac-alu-250) referred as “alum”, ultrapure LPS from E. coli serotype O55:B5 (Cat. No. Tlrl-pb5lps) for in vivo experiments, mouse TLR1–9 Agonist Kit (Cat. No. tlrl-kit1mw), nigericin (Cat. No. tlrl-nig) all from Invivogen (Toulouse, France); BD DIFCO fluid thioglycolate medium (Cat. No. 225650; BD, Argenta, Poznan, Poland); TMB substrate reagent set (Cat. No. 555214; BD, Warsaw, Poland), streptavidin PE (Cat. No. 554061; BD, Warsaw, Poland); LPS from E. coli serotype EH100 (Cat. No. ALX-581-010-L001; Enzo, Farmingdale, USA) for in vitro experiments; murine recombinant IFN-γ (Cat. No. 485-MI; R&D Systems, Bio-Techne, Warsaw, Poland); Peprotech murine recombinant IL-4 (Cat. No. 214-14), Peprotech murine recombinant M-CSF (Cat. No. 315-02), Gibco DMEM with high glucose (Cat. No. 11965092), penicillin-streptomycin (Cat. No. 15070063), Gibco heat-inactivated fetal bovine serum (Cat. No. 10500064), eBioscience Intracellular Fixation Buffer (Cat. No. 00-8222-49), eBioscience Foxp3/Transcription Factor Fixation/Permeabilization concentrate and diluent (Cat. No. 00-5521-00), eBioscience permeabilization buffer (10X) (Cat. No. 00-8333-56) all from Thermo Fisher Scientific (Life Technologies, Warsaw, Poland); inhibitors: JNK-IN-8 (Cat. No. HY-13319), SP600125 (Cat. No. HY-12041), PD98059 (Cat. No. HY-12028) and SB203580 (Cat. No. HY-10256) all from MedChemExpress (Sollentuna, Sweden); PBS (Cat. No. P04-36500; PAN-Biotech, Immuniq, Zory, Poland); sodium chloride injection solution (Cat. No. 3117301, Polpharma, Starogard Gdanski, Poland); BSA (Cat. No. ALB004; BioShop, Epro Science, Wladyslawowo, Poland); Tween-20 (Cat. No. P9416), saponin (Cat. No. 84510), gelatin type A from porcine skin (Cat. No. G-2625) all from Sigma-Aldrich (Merck Life Science, Poznan, Poland); Triton X-100 (Cat. No. 841810492; Poch SA, Gliwice, Poland); 2x Laemmli sample buffer (Cat. No. 1610737; Bio-Rad, Warsaw, Poland); WesternBright ECL HRP substrate (Cat. No. K-12045-D20, Advansta, San Jose, California, USA); paraformaldehyde (Cat. No. 43368.9M; VWR International, Gdansk, Poland); Zombie Aqua Fixable Viability Kit (Cat. No. 423102; Biolegend, Amsterdam, The Netherlands). .. The following antibodies were used: anti-CD3 PerCPCy5.5 (0.1 μg/100 μl; clone 17A2, Cat. No. 100218), anti-F4/80 Alexa Fluor 647 (0.25 μg/100 μl; clone BM8, Cat. No. 123122), anti-CD45 APCCy7 (0.2 μg/100 μl; clone 104; Cat. No. 109824), anti-Ly6C PE-Cy7 (0.005 μg/ml; clone HK1.4, Cat. No. 128017), anti-Ly6G FITC (0.25 μg/100 μl; clone 1A8, Cat. No. 127605) all from Biolegend (Amsterdam, The Netherlands); anti-CD11b BV650 (0.04 μg/100 μl; clone M1/70; Cat. No. 563402), anti-CD11c BV421 (0.2 μg/100 μl; clone HL3; Cat. No. 562782), anti-Ly6G BV711 (0.2 μg/100 μl; clone 1A8; Cat. No. 563979), anti-SiglecF PerCPCy5.5 (0.1 μg/100 μl; clone E50-2440; Cat. No. 565526) all from BD (Warsaw, Poland); anti-CD16/CD32 monoclonal antibodies (0.25 μg/100 μl; clone 93; Cat. No. 14-0161-85), eBioscience anti-c-kit FITC (0.25 μg/100 μl; clone 2B8; Cat. No. 11-1171-85), anti-CD11c PerCPCy5.5 (0.1 μg/100 μl; clone N418, Cat. No. 45-0114), anti-CD19 PerCPCy5.5 (0.1 μg/100 μl; clone 1D3, Cat. No. 45-0193-82), anti-MHC II Alexa Fluor 700 (0.02 μg/100 μl; clone M5/114.15.2, Cat. No. 56-5321-80), anti-SiglecF eFluor660 (0.04 μg/100 μl; clone 1RNM44N, Cat. No. 50-1702-82) all from Thermo Fisher Scientific (Life Technologies, Warsaw, Poland); anti-mouse SLPI – biotin (0.067 μg/100μl; Cat. No. BAF-1735), anti-mouse SLPI (2 μg/ml; Cat. No. AF1735), anti-mouse MMP9 (0.25 μg/ml; Cat. No. AF909-SP) all from R&D Systems (Bio-Techne, Warsaw, Poland); anti-β-actin (1:5000 dilution; clone AC15; Cat. No. A1978), rabbit anti-goat IgG antibody HRP conjugated (0.5 μg/ml; Cat. No. AP106P) all from Sigma-Aldrich (Merck Life Science, Poznan, Poland); StarBright Blue 520 goat anti-mouse IgG (1:2500 dilution; Cat. No. 12005867, Bio-Rad, Warsaw, Poland).
Membrane:Article Title: Tbc1d10c is a selective, constitutive suppressor of the CD8 T-cell anti-tumor response
Article Snippet: .. MC38 cells were pre-incubated with 10 ng/ml murine recombinant Ifn-γ (R&D Systems) and OVA257-264 peptide for 24 hrs and subsequently stained with PKH26 membrane dye as per manufacturer’s protocol (Sigma Aldrich). ..
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