The IVIG preparation (Privigen, CSL Behring AG, Bern, Switzerland) used in this work is a 10% protein solution containing > 98% IgG, and < 0.025 mg/ml IgA. IgG molecules in Privigen are stabilized with 250 mmol/L l-proline (vehicle). Lyophilized, plasma-derived human C1 s esterase inhibitor, C1-INH, (Berinert, CSL Behring GmbH, Marburg, Germany) was reconstituted with 10 ml of sterile water to achieve a final C1-INH dose of 500 U in vehicle solution containing 10 mg/ml of glycine, 8.5 mg/ml of NaCl and 3 mg/ml of sodium citrate. Reconstituted C1-INH was kept at +4 °C and used within 14 days. Both IVIG and C1-INH were administered by slow infusion into the femoral vein either 30 min before (pre-treatment) or at designated time points (1 h and 3 h) after MCAO (post-treatment). Vehicle solutions for both therapeutics (as described above) were infused into control animals at the volume that equaled the volume in which optimal doses of IVIG and C1-INH were delivered.
C1 inh
Manufactured by CSL Behring
Sourced in Germany
C1-INH is a laboratory equipment product used to measure the level of C1 esterase inhibitor, a protein involved in the complement system. It provides a quantitative assessment of C1-INH levels, which is useful for the diagnosis and monitoring of certain medical conditions.
Automatically generated - may contain errors
Lab products found in correlation
Berinert, by CSL Behring (1 mentions)
Alamar blue solution, by Thermo Fisher Scientific (1 mentions)
96 well microplate, by Corning (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Recombinant human wnt3a, by R&D Systems (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
Ang 2, by Fujifilm (1 mentions)
Clodronate disodium, by Merck Group (1 mentions)
Tacs 2 tdt fluorescein kit, by Bio-Techne (1 mentions)
Recombinant mouse il 4, by Thermo Fisher Scientific (1 mentions)
Tamoxifen, by Merck Group (1 mentions)
Pkf115 584, by Novartis (1 mentions)
Anti ly6g antibody, by BioXCell (1 mentions)
Il 1ra, by Merck Group (1 mentions)
Igg isotype control antibody, by BioXCell (1 mentions)
Il 1β, by Thermo Fisher Scientific (1 mentions)
C57bl 6j b6, by Jackson ImmunoResearch (1 mentions)
Il 1β, by Merck Group (1 mentions)
Privigen, by CSL Behring (1 mentions)
Cd90.2 magnetic beads, by Miltenyi Biotec (1 mentions)
7 protocols using c1 inh
1
IVIG and C1-INH Administration in MCAO
2
Perfusion-Induced Endothelial Activation
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After two days of pulsatile flow, cell culture medium was replaced with normal human serum (NHS) 1:10 diluted in 4% dextran DMEM without supplements. PAEC were perfused for different periods of time (10 min, 30 min, 60 min, 120 min). The perfusate (1:10 diluted NHS in 4% dextran DMEM with or without complement inhibitors) was present in 15 ml reservoir tubes (Nalge NUNC) and perfusion was performed in a closed circuit so that the perfusate was recirculated. Usually 10 ml of perfusate were used, but for some experiments the amount was varied from 3 to 10 ml, with a control of static incubation with 200 µl. Four groups were made: Group 1: NHS alone, Group 2: NHS + 10 IU/ml C1 INHibitor (C1 INH, Berinert, provided by CSL Behring, Marburg, Germany), Group 3: NHS + 0.25 mg/ml APT070 (a recombinant, membrane-targeted complement inhibitor based on complement receptor 1, provided by Richard Smith, King’s College, London, UK), Group 4: NHS + 0.3 mg/ml low molecular weight dextran sulfate (DXS, Mw ~5000, provided by Tikomed, Viken, Sweden). For each group, experiments with 3–5 channels were performed. Finally, perfusate was collected and stored at −80 °C. EC in the microchannels were used for immunofluorescence staining.
3
Complement Inhibitors for Traumatic Brain Injury
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A single dose of C1-Inh (15.0 IU; Berinert®; CSL Behring GmbH, Marburg, Germany) or 0.9% saline solution was administered, intravenously (i.v.), by the tail vein 1 h after the CCI injury; CR2-Crry (10 mg/kg) or phosphate-buffered saline (PBS) were administered, i.v., by the tail vein at 1 and 7 h post-CCI; and PMX205 (1 mg/kg, synthesized in-house19 (link)) or 5% glucose vehicle solution were administered by i.p. injection at twice-daily intervals, commencing 1 h post-TBI over a 10-day period. The i.v. injections were performed with mice under isoflurane anaesthesia, and the i.p. injections were administered with mice restrained. Doses and administration times were based on the available literature documenting the effectiveness of each complement inhibitor, or the known half-life of the agent.10 (link),14 (link),20 (link) Control mice received the equivalent amount of vehicle at each dosing interval.
4
AQP4-Expressing Cell Cytotoxicity Assay
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M23-AQP4-expressing CHO cells were plated onto 96-well microplates (Costar, Corning Inc., Corning, NY) at 25,000 cells/well and grown for 24 h. Cells were washed with phosphate-buffered saline (PBS) and incubated at room temperature for 60 min with NMO-IgG or purified IgG from NMO patient serum in live cell buffer (PBS containing 6 mM glucose and 1 mM sodium pyruvate) containing pooled normal human complement serum (Innovative Research, Novi, MI) with or without C1-inh (Berinert, CSL Behring, King of Prussia, PA) in a final volume of 50 µl. Some experiments were done with rat serum, prepared from freshly obtained rat blood and then frozen, in place of the human complement serum. In some experiments, a solution containing pooled normal human complement serum was incubated with C1-inh for specified times before addition to the CHO cells. To measure cytotoxicity, cells were washed twice with PBS, and incubated with 50 µl of a 20% Alamar Blue solution (Invitrogen) for 45 min at 27°C. Cytotoxicity was measured from resorufin fluorescence using a TECAN Infinite M1000 plate reader (TECAN Groups Ltd, Mannedorf, Switzerland) (excitation/emission 560/590 nm).
5
Investigating Wnt Signaling and Inflammation
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BrdU, tamoxifen and clodronate disodium were purchased from Sigma. Human complement C1q and C1 complexes were from Calbiochem. AngII and hydralazine hydrochloride were from Wako. C1-INH (Berinert) was from CSL Behring. PKF115-584 (ref. 22 (link)) was from Novartis. Human recombinant Wnt3A was from R&D. Mouse recombinant IL-4 was from PeproTech. Mouse monoclonal antibody (14/Beta-Catenin) against β-catenin was from BD Transduction Laboratories (immunofluorescence (IF) dilution 1:200). Rat monoclonal antibody (clone CI:A3-1) against mouse F4/80 (IF dilution 1:50), rabbit monoclonal antibody (clone E247) against β-catenin (WB dilution 1:2,000), rabbit polyclonal antibody against axin2 (WB dilution 1:2,000, IF dilution 1:100) and rat monoclonal antibody against BrdU (clone BU1 75(ICR1)) (IF dilution 1:200) were from Abcam. Mouse monoclonal antibody (clone 8E7) against ABC was from Millipore (WB dilution 1:1,000). rabbit polyclonal antibody against actin and alpha-smooth muscle actin (αSMA) were from Sigma (IF dilution 1:200). TACS 2TdT Fluorescein Kit was from Trevigen. Mouse monoclonal antibodies against C1r (WB dilution 1:250) and C1s (WB dilution 1:250) were from R&D. Secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 546 were from Molecular Probes (IF dilution 1:200).
6
Targeting Complement and Cytokines in Lung Transplantation
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Male wild-type C57BL/6J (B6), Fcrγ–/–, Il1r–/–, C1qa–/–, C3–/–, Mbl–/–, and Nr4a–/– mice were obtained from The Jackson Laboratory. IL-1RA (0.2 ng/g body weight i.v.; Sigma-Aldrich) was used for IL-1β–IL-1R antagonism. C1INH (0.4 U/g body weight, Berinert, CSL Behring) and LNP023 (Factor B inhibitor, 30 μg/g body weight, Adooq Bioscience) were injected i.v. in recipients 24 hours before and 1 hour after lung transplantation. IL-1β (10 μg/kg, i.v.; Thermo Fisher Scientific) was injected 8 hours prior to LRA injection. Neutrophils were depleted by using anti-Ly6G antibody (12.5 mg/kg body weight; Bio X Cell, clone 1A8). Complement C5 was inhibited by using a C5-blocking antibody (100 μg/mouse, 1 hour before lung transplant; provided by Alexion Pharmaceuticals). Control mice were treated with the same amounts of IgG isotype control antibody (Bio X Cell). All mice were maintained in a specific pathogen–free facility at the Center for Comparative Medicine at Northwestern University and used for the described experiments at the age of 9–14 weeks and between 24 and 28 g of body weight.
7
GVHD Model with Mannose-Binding Lectin Deficiency
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