Orthotopic injection was performed between the second and third mammary gland of adult female mice (age range 8-12 weeks) with either 1x106 LM2, 1x106 BR16 or 0.25x106 4T1 cells, expressing the fluorescent construct GFP/Luc or mCherry/Luc. Cells were inoculated in 50% Cultrex Path Clear Reduced Growth Factor Basement Membrane Extract (R&D Biosystems, 3533-010-02) and 50% PBS. In mice injected with cells carrying a dox-inducible construct, water containing 0.5 mg/ml Dox (Sigma, D9891-25G) and 5% sucrose (Sigma, S9378) was administered 3 times a week upon tumor formation and for a maximum of 3 months. Injection of 0.02 mg in PBS of recombinant mEphrin-B2-hFC chimera or ChromPure IgG hFC fragment was performed intra-peritoneal (i.p.) and with a frequency of twice per week. Injection of 25 mg/Kg Bevacizumab in PBS (Genentech, Avastin®) or 26.25 mg/Kg Paclitaxel in PBS (Bristol-Myers Squibb, Taxol®) or Ultra-LEAF purified human IgG1 Isotype control (Biolegend, 403502) was performed i.p. and with a frequency of twice per week. Injection of 15 mg/Kg Paclitaxel in PBS (Bristol-Myers Squibb, Taxol®) was performed i.p. once a week. Bevacizumab and Paclitaxel were obtained from the Pharmacy of the University Hospital Basel, under permit #RL0004-V07-B02.
Ultra leaf purified human igg1 isotype control
Manufactured by BioLegend
The Ultra-LEAF purified human IgG1 Isotype control is a lab equipment product. It serves as a control reagent for flow cytometry experiments involving human IgG1 antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Sucrose, by Merck Group (1 mentions)
Cultrex path clear reduced growth factor basement membrane extract, by R&D Systems (1 mentions)
Taxol, by Bristol-Myers Squibb (1 mentions)
Elisa plate, by Corning (1 mentions)
Hrp labeled streptavidin, by Thermo Fisher Scientific (1 mentions)
Human il 6, by Miltenyi Biotec (1 mentions)
Human m csf, by Thermo Fisher Scientific (1 mentions)
Stattic, by Bio-Techne (1 mentions)
Tofacitinib, by Selleck Chemicals (1 mentions)
Roactemra, by Roche (1 mentions)
Sunitinib, by Pfizer (1 mentions)
3 protocols using ultra leaf purified human igg1 isotype control
1
Orthotopic Tumor Establishment and Treatment
2
Recombinant Protein Binding Assay
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For recombinant proteins an ELISA-based binding assay was implemented measuring the interactions of hVISTA-Fc fusion protein with plate-bound human receptor proteins at pH 6.0 and pH 7.4. hVSIG-3-Fc (Acro Biosystems VS3-H5258), hVSIG-8-Fc (9200-VS), hPSGL-1-Fc (3345-PS), hSyndecan-2 (2965-SD) and hLRIG-1 (8504-LR; all R&D Systems) were coated in ELISA plates (Corning) at 5 μg/ml. Plates were blocked in 1% NFDM in PBS at pH 6.0 or 2% BSA in PBS at pH 7.4. For binding assays, C-terminal biotinylated hVISTA-Fc (R&D Systems AVI7126-050) was serially diluted in 1% NFDM in PBS at pH 6.0 or 2% BSA in PBS at pH 7.4 before being added to blocked plates. For the competition assay, SNS-101 or isotype control (Ultra-LEAF Purified Human IgG1 Isotype Control, BioLegend 403501) were serially diluted in 1% NFDM in PBS at pH 6.0 and pre-incubated with 1 μg/ml biotinylated hVISTA-Fc at pH 6.0 for 2 h at room temperature. After washing in PBS containing 0.05% Tween 20 at pH 6.0 or pH 7.4, HRP-labeled streptavidin (Thermo Fisher Scientific 21134) diluted 1:200 in PBS-NFDM at pH 6.0 or PBS-BSA at pH 7.4 was used as detection reagent.
3
Modulation of IL-6 and M-CSF Signaling
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Stattic (BioTechne, 2789) and Tofacitinib (Selleckchem, S2789) were dissolved in DMSO. To inhibit IL-6 signaling, cells were pre-incubated with 1 μM Stattic or Tofacitinib for 1 h at 37°C and 5% CO2. After pre-incubation, 25 ng/mL human IL-6 (Miltenyi), 25 ng/mL human M-CSF (PeproTech), or BLM-CM (final concentration 50%) was added to the cells where indicated. After a 48 h culture period, cells were harvested and analyzed by flow cytometry. Sunitinib (Sutent, Pfizer) was suspended in DMSO to obtain a 1.43 mM stock solution. To inhibit IL6R and CSF1R signaling, cells were pre-incubated with 10 μg/mL tocilizumab (RoActemra, Roche) and/or 100 nM Sunitinib, or 10 μg/mL Ultra-LEAF Purified anti-human M-CSF antibody (Clone A16067H, Biolegend) for 20 min at 37°C and 5% CO2. Control samples were incubated with 10 μg/mL mouse IgG2b Isotype control or 10 μg/mL Ultra-LEAF Purified Human IgG1 Isotype control (Clone QA16A12, Biolegend) and/or DMSO as vehicle control. After pre-incubation, 1 ng/mL human IL-6 (Miltenyi), 10 ng/mL human M-CSF (PeproTech), or 50% BLM-CM was added to the cells where indicated. After a 48 h culture period, cells were harvested and divided for phenotype analysis by flow cytometry or re-seeded for allogeneic T cell proliferation assays.
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