To evaluate the therapeutic effects of mAbs against cytokines, we inoculated a lethal dose of CVA2 into 5-day-old mice (i.m.). After 12 h, these mice were injected i.p. with Ultra-LEAFTM purified neutralizing monoclonal anti-mouse IL-6, TNF-α, and MCP-1 IgG mAb (Biolegend) at 1.33 mg/kg (Khong et al., 2011 (link)), 50 mg/kg TNF-α (Scanga et al., 1999 (link); Via et al., 2001 (link)), and 10 mg/kg MCP-1 (Morrison et al., 2003 (link)) per mouse. Suckling mice from the control group were injected with equivalent purified rat IgG1 (isotype control, Biolegend). All suckling mice were monitored daily upon infection for the occurrence of clinical symptoms and mortality up to day 15 post-infection as the experimental endpoint.
Rat igg1
Manufactured by BioLegend
Sourced in United States
Rat IgG1 is an isotype control antibody derived from the rat species. It serves as a control for experiments involving rat-derived antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Mouse igg1, by BioLegend (3 mentions)
Anti il 6 antibody, by BioLegend (2 mentions)
Streptavidin alexa 633, by Thermo Fisher Scientific (1 mentions)
Accuri c6 cytometer, by BD (1 mentions)
Ebiomm17f3, by Thermo Fisher Scientific (1 mentions)
Anakinra, by BioVitrum (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Thermo Fisher Scientific (1 mentions)
Soluble anti cd3, by BD (1 mentions)
Maz51, by Merck Group (1 mentions)
Mouse igg2b, by BioLegend (1 mentions)
Rat igg2b, by BioLegend (1 mentions)
Mouse igg2a, by BioLegend (1 mentions)
Rat igg2a, by BioLegend (1 mentions)
Dnase, by Merck Group (1 mentions)
Liberase, by Merck Group (1 mentions)
Mp5 20f3, by BioLegend (1 mentions)
B6 sjl ptprca pepcb boyj mice, by Jackson ImmunoResearch (1 mentions)
B6 129s6 gt rosa 26sortm9 cag tdtomato hze j mice, by Jackson ImmunoResearch (1 mentions)
14 protocols using rat igg1
1
Evaluating Anti-Cytokine mAb Therapy against Lethal CVA2 Infection
2
Phenotyping mouse CAR T cells
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Mouse CAR T cells were stained with antibodies for CD3ε (145-2c11 Biolegend), CD4 (Clone GK1.5 Biolegend), and CD8α (clone 53-6.7 Caltag) as previously described [40 (link)]. CAR surface expression was assessed with either biotinylated Protein L (catalog no. 29997 Pierce) followed by streptavidin-Alexa 633 (Life Technologies/Invitrogen) or with a F(ab’)2-labeled with phycoerythrin (PE) (catalog no. 115-116-146 Jackson Immuno). Expression of T-bet was assessed with a T-bet-specific antibody (clone 4B10 Biolegend), in conjunction with a FoxP3/Transcription Factor Buffer Staining Kit (ebioscience) according to the manufacturer’s protocol. Rat IgG1 (Clone RTK2071 Biolegend) was used as the isotype control for intracellular staining. Samples were analyzed on an Accuri C6 Cytometer (BD Biosciences, San Jose, CA, USA).
3
Silica Particle Exposure Model
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Natural crystalline silica particles (Min-U-Sil 5 ground silica; size distribution: 97% <5 μm diameter, 80% <3 μm diameter; median diameter 1.4 μm) were obtained from the U.S. Silica Company (Frederick, MD, USA) [10 (link)]. Silica particles were boiled in 1 N HCl to remove endotoxins. Before use, the suspension was autoclaved and then sonicated as described previously [11 ]. To block IL-17, mice were treated with antimouse IL-17 neutralizing mAb (clone eBioMM17F3; eBioscience, San Diego, CA, USA) 100 μg intraperitoneally (i.p.)once a week since 1 day before silica exposure until killed. Control mice were administered rat IgG1 (BioLegend, San Diego, CA, USA) once a week since 1 day before silica exposure until killed [12 (link)]. To block the IL-1β receptor IL-1RI, mice were administered anakinra (1 mg; Biovitrum, Stockholm, Sweden) in sterile saline (i.p. 100 μl in total) twice daily since 1 day before silica exposure until killed [13 (link)]. Control mice were administered sterile saline alone (i.p. 100 μl in total) twice daily since 1 day before silica exposure until killed.
4
Proliferation and Suppression Assays for T-cells
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For the proliferation assay, one day after OVA challenge, cell suspensions were generated from the pooled LN and spleens from individual mice as described above. Cells were incubated in RPMI-1640 containing 10 % FCS, 2 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, and 1.25 mg/mL amphotericin B (all Gibco BRL, CA, USA) (complete medium) in the presence of 1, 10, and 100 mg/mL OVA at 37 °C in 5 % CO2. Cell proliferation was evaluated by [3H] thymidine (3H-TdR) incorporation. Cytokine content was analysed in culture supernatants by ELISA from Bender Med Systems, Vienna, Austria.
For suppression assays, 1 × 105 CD4+CD25− T-cells/well, 5 × 104 CD4+CD25+ T-cells/well, or both populations were cultured in 96-well U-bottom plates with 1 × 105 APCs/well for 72 h at 37 °C in complete RPMI 1640 medium (0.2 mL/well) in triplicate. Cultures were stimulated with 1 μg/mL soluble anti-CD3 (BD PharMingen, San Diego, CA, USA) with or without 0.1 μg/mL SJMHE1. Certain wells were added with 3 μg/mL rat IgG1 anti-mouse IL-10 (Biolegend Inc., San Diego, CA, USA), 0.5 μg/mL rat IgG1 anti-mouse TGF-β1 (US Biological, Swampscott, MA, USA), or 3 μg/mL rat IgG1 (Biolegend). Proliferation was assessed by incubation with 0.5 μCi/well 3H-thymidine and measuring the incorporation during the final 16 h of culture.
For suppression assays, 1 × 105 CD4+CD25− T-cells/well, 5 × 104 CD4+CD25+ T-cells/well, or both populations were cultured in 96-well U-bottom plates with 1 × 105 APCs/well for 72 h at 37 °C in complete RPMI 1640 medium (0.2 mL/well) in triplicate. Cultures were stimulated with 1 μg/mL soluble anti-CD3 (BD PharMingen, San Diego, CA, USA) with or without 0.1 μg/mL SJMHE1. Certain wells were added with 3 μg/mL rat IgG1 anti-mouse IL-10 (Biolegend Inc., San Diego, CA, USA), 0.5 μg/mL rat IgG1 anti-mouse TGF-β1 (US Biological, Swampscott, MA, USA), or 3 μg/mL rat IgG1 (Biolegend). Proliferation was assessed by incubation with 0.5 μCi/well 3H-thymidine and measuring the incorporation during the final 16 h of culture.
5
Blocking VEGF-C and IL-6 Signaling in Vessels
6
Comprehensive Flow Cytometry Antibody Panel
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The antibodies for flow cytometry experiments: anti-mouse CD3; anti-mouse CD4; anti-mouse CD8a; anti-mouse CD25; anti-mouse CD38; anti-mouse CD69; anti-mouse CD70; anti-mouse CD11b; anti-mouse CD11c; anti-mouse CD19; anti-CD62L; anti-mouse CD80; anti-mouse CD86; anti-mouse CD68; anti-mouse F4/80; anti-mouse TNFα; anti-Granzyme B; anti-mouse/human CD44; anti-mouse CD183 (CXCR3); anti-mouse CD184 (CXCR4); anti-mouse CD185 (CXCR5); anti-mouse CD186 (CXCR6); anti-mouse CD1d; anti-mouse CD120a (TNFRSF1a); anti-mouse CD152 (CTLA-4); anti-mouse CD279 (PD-1); anti-mouse CD274 (B7-H1 or PD-L1); anti-mouse CD273 (B7-DC or PD-L2); anti-mouse CD117 (c-Kit); anti-mouse Ly-6A/E (SCA-1); anti-mouse Lineage Cocktail with Isotype Ctrl; mouse IgG1, κ Isotype; mouse IgG2b, κ Isotype Ctrl; mouse IgG2a, κ Isotype; rat IgG2b, κ Isotype; Armenian Hamster IgG Isotype; Syrian Hamster IgG Isotype; rat IgG2a, κ Isotype and rat IgG1, κ Isotype Ctrl were procured from BioLegend. The anti-mouse CD34, anti-perforin 1 antibodies and regulatory T cells (Treg) staining kit were purchased from eBioscience.
7
Isolation and Culture of Colonic Fibroblasts
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The protocol for the isolation of colonial fibroblasts (CF) was modified according to Roulis et al (2014 (link)). Briefly, colons were isolated from mice, flushed with PBS, and then opened longitudinally and processed as follows. Intestinal pieces were cut into small pieces and incubated in EDTA‐containing buffer (HBSS/2% FCS/5 mM EDTA) at 37°C for 20 min under continuous shaking to release colonic epithelial cells (CEC). After extensive washes, the tissue was further processed by treatment with 62.5 μg/ml Liberase and 40 μg/ml DNAse (Sigma) in HBSS/2% FCS for 50 min at 37°C under continuous shaking to isolate CF. Cells were filtered through a 70‐μm strainer, washed with PBS, and subsequently analyzed or taken in culture. CF was cultured in DMEM/10%FCS for 2 passages (2P‐CF). To induce the formation of PC, 70–90% confluent 2P‐CF was cultured for 24 h in a starvation medium containing 1% BSA. In specific experiments, cells were cultured in the presence of inflammatory (inf) or control (co) conditioned medium (CM). To obtain those, colons were isolated from control mice and animals treated for 7 days with DSS, washed with PBS, and subsequently took in culture for 4 h in the medium. For blocking experiments, 5 μg/ml of antagonistic anti‐IL‐6 antibody MP5‐20F3 and isotype control (rat IgG1) were used (both Biolegend).
8
Adoptive Transfer and In Vivo Modulation of Th17 Cells in EAE
9
Adoptive Transfer of CFSE-labeled DLI
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Recipient mice were injected intraperitoneally (i.p.) with 200 µg of depleting antibodies diluted in 100 𝜇L of PBS to CD4 (GK1.5, BioXCell) and CD8 (2.43, BioXCell), respectively.
The injection was performed every third day, starting on one day after adoptive transfer of CFSE-labeled DLI (24) . Control mice received equal amount of isotope control antibodies (Rat IgG1, BioLegend) or equal volumes of PBS.
The injection was performed every third day, starting on one day after adoptive transfer of CFSE-labeled DLI (24) . Control mice received equal amount of isotope control antibodies (Rat IgG1, BioLegend) or equal volumes of PBS.
10
Anti-Inflammatory Therapy for EAE Mouse Model
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Glycolipid AA2 and D-sphingosine were initially dissolved in DMSO, followed by dilution with PBS and brief sonication. After EAE induction, mice were injected i.p. every other day with 10 μg AA2/mouse or with vehicle. Daily injection of D-spingosine via i.p. or i.v. started on day 3 after EAE induction. Anti-IL-17 neutralizing antibody (100 μg/mouse, Biolegend) or its isotype control (Rat IgG1) was given at Day 3, 5, 7, 9, and 11 after EAE induction. All EAE mice were euthanized on day 20–25. Spinal cords and brains were fixed by 4% paraformaldehyde (PFA) for immunohistochemistry (IHC) and immunofluorescence staining.
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