toxinsensor gel clot endotoxin assay kit, by GenScript - Product details

1

Recombinant Peroxiredoxin Protein Expression

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Prx (XP_648522.1) and its three fragments (Fragment-1: from 1 to 100 amino acids; Fragment-2: from 71 to 170 amino acids; Fragment-3: from 134 to 233 amino acids) were amplified from E. histolytica cDNA using primers listed in Table 1. The gene fragment was inserted into a pET19b vector (Novagen, Germany) and transformed into E. coli BL21 (DE3) pLysS for protein expression. Mutant Prx was also cloned using primers listed in Table 1. Based on nucleotide sequence of Eh-Prx (XM_643430.2), the codon of cysteine in both cysteine active sites of Eh-Prx (from 5’ to 3’, at sites of 264 and 627, respectively) was mutated from UGU to GGU, resulting in mutation from cysteine to glycine. Recombinant protein was filtered through a 0.2 μm filter, while endotoxin levels of the recombinant protein were detected using a Toxin Sensor™ Gel Clot Endotoxin Assay Kit (Genscript, L00351, USA).

Li X., Feng M., Zhao Y., Zhang Y., Zhou R., Zhou H., Pang Z., Tachibana H, & Cheng X. (2021). A Novel TLR4-Binding Domain of Peroxiredoxin From Entamoeba histolytica Triggers NLRP3 Inflammasome Activation in Macrophages. Frontiers in Immunology, 12, 758451.

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2

Versikine Production and MutuDC1940 Activation

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5x10⁶ HEK293 and HEK-Vkine expressing cells (a kind gift of Dr. Suneel S. Apte, Cleveland Clinic Lerner Research Institute) were seeded in T-175 cell culture flasks and cultured in DMEM 10% FBS media. After 75 to 80% confluency, the cell media was changed to DMEM 1% FBS media. Subsequently, media supernatant was collected after 48 hours of incubation. The collected supernatant was centrifuged to remove debris and filtered with 0.45μ filter. The filtered supernatant was then concentrated 30 times to the initial volume using Sartorius Vivaspin 20, 10,000 MWCO PES concentrator (Cat. No. VS2001). Endotoxin assay was performed using Genscript ToxinSensor Gel Clot Endotoxin Assay Kit (Cat. No. L00351) according to the manufacturer’s instructions to rule out contamination. The presence of versikine in concentrated supernatant was confirmed using western blot using c-Myc Antibody (Novus Bio-c-Myc Antibody (9E10) - Chimeric NBP2-52636). Concentrated supernatant containing versikine was then used to treat MutuDC1940 cells. 2 × 10⁵ MutuDC1940 cells per well were seeded in 12 well plate. The following day, 10% supernatant was added to the plate media. Cells were then incubated for 72 hours. After incubation, total RNA was extracted from MutuDC1940 cells.

Papadas A., Deb G., Cicala A., Officer A., Hope C., Pagenkopf A., Flietner E., Morrow Z.T., Emmerich P., Wiesner J., Arauz G., Bansal V., Esbona K., Capitini C.M., Matkowskyj K.A., Deming D.A., Politi K., Abrams S.I., Harismendy O, & Asimakopoulos F. (2022). Stromal remodeling regulates dendritic cell abundance and activity in the tumor microenvironment. Cell reports, 40(7), 111201.

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3

Versikine Production and MutuDC1940 Activation

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5x10⁶ HEK293 and HEK-Vkine expressing cells (a kind gift of Dr. Suneel S. Apte, Cleveland Clinic Lerner Research Institute) were seeded in T-175 cell culture flasks and cultured in DMEM 10% FBS media. After 75 to 80% confluency, the cell media was changed to DMEM 1% FBS media. Subsequently, media supernatant was collected after 48 hours of incubation. The collected supernatant was centrifuged to remove debris and filtered with 0.45μ filter. The filtered supernatant was then concentrated 30 times to the initial volume using Sartorius Vivaspin 20, 10,000 MWCO PES concentrator (Cat. No. VS2001). Endotoxin assay was performed using Genscript ToxinSensor Gel Clot Endotoxin Assay Kit (Cat. No. L00351) according to the manufacturer’s instructions to rule out contamination. The presence of versikine in concentrated supernatant was confirmed using western blot using c-Myc Antibody (Novus Bio-c-Myc Antibody (9E10) - Chimeric NBP2-52636). Concentrated supernatant containing versikine was then used to treat MutuDC1940 cells. 2 × 10⁵ MutuDC1940 cells per well were seeded in 12 well plate. The following day, 10% supernatant was added to the plate media. Cells were then incubated for 72 hours. After incubation, total RNA was extracted from MutuDC1940 cells.

Papadas A., Deb G., Cicala A., Officer A., Hope C., Pagenkopf A., Flietner E., Morrow Z.T., Emmerich P., Wiesner J., Arauz G., Bansal V., Esbona K., Capitini C.M., Matkowskyj K.A., Deming D.A., Politi K., Abrams S.I., Harismendy O, & Asimakopoulos F. (2022). Stromal remodeling regulates dendritic cell abundance and activity in the tumor microenvironment. Cell reports, 40(7), 111201.

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4

Preparation and Use of zVAD and sFasL

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The broad caspase inhibitor zVAD was obtained from Bachem, Basel, Switzerland. A stock preparation of 20 mg/mL was prepared in sterile DMSO to solubilize the compound. On the day of the experiment, the stock was diluted in PBS to a final DMSO concentration of 5%. For control experiments, a solution containing 5% DMSO in PBS (vehicle), was used. The endotoxin concentration in the dissolved and diluted zVAD was below 0.25 EU/mL (assay lower limit) as determined by a ToxinSensor Gel Clot Endotoxin Assay Kit (GenScript, Piscataway, NJ).
The soluble, human, recombinant SuperFas Ligand (rh-sFasL) was obtained from Enzo Life Sciences (Raamsdonksveer, the Netherlands).

van den Berg E., Bal S.M., Kuipers M.T., Matute-Bello G., Lutter R., Bos A.P., van Woensel J.B, & Bem R.A. (2015). The caspase inhibitor zVAD increases lung inflammation in pneumovirus infection in mice. Physiological Reports, 3(3), e12332.

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5

Purification of Echinococcus Antigen B

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AgB was purified from E. granulosus s.s. ECF according to a previously published method [13 (link)]. In brief, after collection from echinococcal cysts, ECF was centrifuged at 10,000×g for 20 min at 4 °C, and the supernatant was filtered through 0.45-μm filter membranes (Millipore). The clarified ECF was used for i.p. injection of mice. To purify AgB, ECF was precipitated by adding sodium acetate at a final concentration of 5 mM, pH 5.0. After centrifugation at 5000×g for 15 min at 4 °C, the sediment was resuspended in PBS and then heated in boiling water for 5 min. After further centrifugation at 10,000×g for 15 min at 4 °C, the supernatant was collected and filtered through a 0.22-μm filter. Endotoxin was removed using endotoxin removal resin (ToxinEraser™ Endotoxin Removal Kit, GenScript Biotech Corp., China). The absence of lipopolysaccharide (LPS) in the supernatant was further verified using gel clot assays (ToxinSensor™ Gel Clot Endotoxin Assay Kit, GenScript Biotech Corp., China). Mice in the AgB-treated group received purified native AgB daily (100 μg/day) by i.p. injection for 5 days.

Bao J., Qi W., Sun C., Tian M., Jiao H., Guo G., Guo B., Ren Y., Zheng H., Wang Y., Yan M., Zhang Z., McManus D.P., Li J, & Zhang W. (2022). Echinococcus granulosus sensu stricto and antigen B may decrease inflammatory bowel disease through regulation of M1/2 polarization. Parasites & Vectors, 15, 391.

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6

Limulus Test for Gram-Negative Endotoxins

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The Limulus test directly detects the endotoxins of Gram-negative bacteria. The test was only carried out on blood samples in which no Gram-negative bacteria were culturally detected, but were detected in the corresponding milk sample. The test was carried out using the ToxinSensor™ Gel Clot Endotoxin Assay Kit (GenScript USA Inc., Piscataway, NJ, USA). The limulus amoebocyte lysate contains a gel-forming protein from blood cells of the Horseshoe crab (Limulus polyphemus), which reacts with the endotoxins of Gram-negative bacteria. Blood samples were considered as positive if the viscosity of the mixture increased. The minimal detection limit of the endotoxin level occurred at 0.25 EU/mL. As this test can only detect endotoxins, a positive result of the blood samples was not defined as bacteremia, but showed an infiltration of lipopolysaccharides into the blood.

Krebs I., Zhang Y., Wente N., Leimbach S, & Krömker V. (2023). Bacteremia in Severe Mastitis of Dairy Cows. Microorganisms, 11(7), 1639.

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7

Purification of Acanthamoeba lugdunensis Proteins

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Acanthamoeba lugdunensis KA/E2 strain, isolated from human cornea inflammation patient in Korea, it was maintained in PYG medium. The KA/E2 strain has the same molecular characteristics as the A. lugdunensis L3A strain (ATCC 50240) [20 (link)]. To obtain total protein, live trophozoites were incubated in PYG medium for one week at 25°C. Following centrifugation at 12,000g for 30 min, the total protein was extracted from the pellet according to protocol of manufacture (Cell lysis, ThermoFisher Scientific Co. Waltham, MA USA). After obtaining total proteins, the ToxinSensor Gel Clot Endotoxin Assay Kit (Gen-Script, Piscataway, New Jersey, USA) was used to eliminate endotoxins.

Song S.M., Kang S.A., Park H.K., Kim D.H., Park S.Y., Jang S.B, & Yu H.S. (2018). Acanthamoeba profilin elicits allergic airway inflammation in mice. PLoS Neglected Tropical Diseases, 12(12), e0006979.

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8

Purification of rAceyCP1, rAceyCPL, and rAceySKPI3

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rAceyCP1, rAceyCPL and rAceySKPI3 were purified from X-33 culture supernatants by immobilized metal affinity chromatography using a Ni resin and column (GenScript). Proteins bound to the resin were washed with Triton X-100 to reduce endotoxin levels to <1 EU/μg. The eluates were buffer exchanged into PBS (pH 7.4) by dialysis, and then filter sterilized with 0.22 μm Millex-GP Syringe Filters. Endotoxin levels were detected by ToxinSensor Gel Clot Endotoxin Assay Kit (GenScript). Protein concentrations were determined by Bradford assay using BSA as standard (GenScript).

Noon J.B., Schwarz E.M., Ostroff G.R, & Aroian R.V. (2019). A highly expressed intestinal cysteine protease of Ancylostoma ceylanicum protects vaccinated hamsters from hookworm infection. PLoS Neglected Tropical Diseases, 13(4), e0007345.

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9

Recombinant Mutant C-Reactive Protein Production

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Recombinant human mCRP (rmCRP) was produced as described previously [26 (link)]. The protein was mutated to replace both cysteine residues with alanine residues (i.e. C36A and C97A) and was further modified using a reverse acylation procedure to enhance aqueous solubility [26 (link), 27 ]. Based on testing using the ToxinSensor™ Gel Clot Endotoxin Assay Kit (GenScript; cat# L00351; Piscataway, NJ, USA), 20 μg/ml mCRP contains less than 0.125 EU/ml (∼0.025 ng/ml) endotoxin levels. Endotoxin test details can be found in the Supplementary materials and methods. The rmCRP recombinant protein will be referred to as ‘mCRP’ for all experiments discussed herein.

Chirco K.R., Whitmore S.S., Wang K., Potempa L.A., Halder J.A., Stone E.M., Tucker B.A, & Mullins R.F. (2016). Monomeric C-reactive protein and inflammation in age-related macular degeneration. The Journal of pathology, 240(2), 173-183.

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10

Purification of Recombinant Proteins PrtA1 and PrtA2

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E. coli BL21 (DE3) was transformed with an expression vector. Protein expression was induced in the exponential phase [optical density (OD) at 600 nm = 1] using 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). At 3 h after induction, the bacteria were harvested and resuspended in lysis buffer [20 mM Tris (pH 8.0), 5 mM imidazole, 500 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1× protease inhibitor cocktail (Roche)], followed by sonication on ice. The recombinant protein PrtA105-His (PrtA1) was purified using Ni-NTA resin (GE Healthcare) according to the manufacturer’s instructions. The other recombinant protein, GST-PrtA105-His (PrtA2), was further purified with glutathione-Sepharose® 4B (Millipore). Lipopolysaccharide (LPS) contamination was reduced by using 0.1% Triton X-114 in the washing step [36 (link)]. The residual LPS was less than 1 EU/μg protein, as determined by a ToxinSensor™ Gel Clot Endotoxin Assay Kit (GenScript).

Hsu C.F., Hsiao C.H., Tseng S.F., Chen J.R., Liao Y.J., Chen S.J., Lin C.S., Sytwu H.K, & Chuang Y.P. (2018). PrtA immunization fails to protect against pulmonary and invasive infection by Streptococcus pneumoniae. Respiratory Research, 19, 187.

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Toxinsensor gel clot endotoxin assay kit

Lab products found in correlation

15 protocols using toxinsensor gel clot endotoxin assay kit

Recombinant Peroxiredoxin Protein Expression

Versikine Production and MutuDC1940 Activation

Versikine Production and MutuDC1940 Activation

Preparation and Use of zVAD and sFasL

Purification of Echinococcus Antigen B

Limulus Test for Gram-Negative Endotoxins

Purification of Acanthamoeba lugdunensis Proteins

Purification of rAceyCP1, rAceyCPL, and rAceySKPI3

Recombinant Mutant C-Reactive Protein Production

Purification of Recombinant Proteins PrtA1 and PrtA2

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