Specific inhibitors described previously were used for blockage of NET formation and have been described previously23 (link),34 (link),40 (link),46 (link),90 (link)–94 (link). The following inhibitors were used: the NE inhibitor Suc-Ala-Ala-Pro-Val chloromethyl ketone (CMK; 1 mM final concentration, Sigma), NADPH oxidase (NOX) inhibitor diphenylene iodonium (DPI; 10 μM final concentration, Sigma), the MPO inhibitor 4-Aminobenzoic acid hydrazide (ABAH; 100 μM final concentration, Sigma), TLR4 signaling inhibitor CLI-095 (1 μg/mL final concentration; InvivoGen), the TLR4 signaling inhibitor polymixin B (100 μg/mL final concentration; InvivoGen, San Diego, CA), the PKC inhibitor Bisindolylmaleimide I (310 nM final concentration, Cayman Chemical, Ann Arbor, MI), and the ROS scavenger inhibitor ascorbic acid (Vitamin C) (200 μM final concentration, Cayman Chemical, Ann Arbor, MI). Cells were pre-incubated with inhibitors for 30 min at 37 °C prior to stimulation as described above23 (link),34 (link).
Polymixin b
Manufactured by InvivoGen
Sourced in United States
Polymixin B is a cationic antimicrobial peptide that disrupts the cell membrane of Gram-negative bacteria. It is commonly used in cell culture applications to prevent bacterial contamination.
Automatically generated - may contain errors
Lab products found in correlation
Diphenyleneiodonium dpi, by Merck Group (1 mentions)
Cli 095, by InvivoGen (1 mentions)
Bisindolylmaleimide 1, by Cayman Chemical (1 mentions)
Suc ala ala pro val chloromethyl ketone cmk, by Merck Group (1 mentions)
4 aminobenzoic acid hydrazide abah, by Merck Group (1 mentions)
Hek blue reporter cells, by InvivoGen (1 mentions)
R d duo set kits, by R&D Systems (1 mentions)
Melatonin, by InvivoGen (1 mentions)
Lipopolysaccharides (lps), by InvivoGen (1 mentions)
4 protocols using polymixin b
1
Inhibition of NETosis Pathways
2
Evaluating Innate Immune Receptor Activation
3
Endotoxin Mitigation in Recombinant OPN Assays
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Human recombinant OPN proteins showed evidence of endotoxin contamination (Pierce LAL assay; Thermo Scientific, Waltham, MA, USA). To inhibit endotoxin effects on assays, we pretreated all samples with polymixin‐B (PMB; InvivoGen, San Diego, CA, USA). This was shown to inhibit the effects of E. coli‐derived lipopolysaccharide (LPS; tested range 10–100 ng ml−1) on human macrophages. Macrophages were pretreated with 100 μg ml filtered PMB sulfate in PBS 30 min prior to stimulation with OPN (InvivoGen). polymixin‐B is a positively charged cyclic polypeptide that binds to and inhibits the actions of the negatively charged and bioactive lipid A tail of endotoxin (Cavaillon & Haeffner‐Cavaillon, 1986 ). The pretreatment of cells with PMB was optimized through testing of IL‐6 and tumour necrosis factor‐α secretion from human macrophages assayed by enzyme‐linked immunosorbent assay ELISA and FACS bead array. Endotoxin‐depleted recombinant human and murine OPN‐a was also obtained for testing and optimization of inflammatory responses (R&D Systems).
For mRNA assays, macrophages were treated with OPN proteins for 4 h. For protein assays, macrophages were treated with OPN proteins for 24 h. For protein assay by ELISA, R&D Duo Set kits were used (R&D Systems).
For mRNA assays, macrophages were treated with OPN proteins for 4 h. For protein assays, macrophages were treated with OPN proteins for 24 h. For protein assay by ELISA, R&D Duo Set kits were used (R&D Systems).
4
Leydig Cell-Macrophage Co-Culture
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Isolation of Leydig cells for culturing was preformed according to a procedure described in [20 (link)]. The co-culture experiment of testicular macrophages and Leydig cells has also been previously described [21 (link),22 (link)]. In the co-culture system, testicular macrophages (0.4 × 105/mL) were cultured in a transwell plate; Leydig cells (1.6 × 105/mL) were cultured in the bottom of the plate (Corning Company, 3491, New York, NY, USA). The medium without or with melatonin (10−7 M), 100 ng/mL LPS and polymixin B (10−6 M) (TLR4 inhibitor) (InvivoGen, San Diego, CA, USA) was added to the Leydig cell culture group and co-culture group, respectively. Each preparation was cultured at CO2 5%, 34 °C. The cells and medium were collected 6h later for further analysis.
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