Highly pure rough LPS from E. coli K12 strain LCD25 was obtained from List biological labs. The FITC labelled LPS from E. coli 014 (Ra) and 3H/14C labelled LPS from Salmonella enterica sv. Typhimurium PR122 (Rc) were kind gift from Prof. Robert Munford (NIAID). Pooled normal human plasma was from Innovative Research. Endotoxin free water was from GE Healthcare Life Sciences. Fluorescein isothiocyanate (FITC), Alexa 488 hydrazide and Alexa 594 NHS were obtained from Molecular Probes. Purified apo-A1 from human plasma was obtained from Athens Research & Technology. The rabbit anti-apoA1 antibody was from Abcam. The TNF-α ELISA kit was from R&D. Chromogenic LAL Endotoxin Assay Kit was from Genscript. Recombinant human LBP protein was from R&D.
Chromogenic lal endotoxin assay kit
Manufactured by GenScript
Sourced in China
The Chromogenic LAL Endotoxin Assay Kit is a laboratory testing product used to detect and quantify endotoxin levels in various samples. It utilizes the Limulus Amebocyte Lysate (LAL) reaction to provide a colorimetric measurement of endotoxin concentration.
Automatically generated - may contain errors
Lab products found in correlation
Orn8l, by Chemgenes (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Anti cd14 magnetic bead, by Miltenyi Biotec (2 mentions)
Orn8l, by GenScript (2 mentions)
Tnf α elisa kit, by R&D Systems (1 mentions)
Pooled normal human plasma, by Innovative Research (1 mentions)
Endotoxin free water, by GE Healthcare (1 mentions)
Alexa 594 nhs, by Thermo Fisher Scientific (1 mentions)
Recombinant human lbp protein, by R&D Systems (1 mentions)
Amicon ultra 15 centrifugal filter, by Merck Group (1 mentions)
Nanoassemblr benchtop instrument, by Precision Nanosystems (1 mentions)
Genvoy ilm, by Precision Nanosystems (1 mentions)
Ni2 nta fast start kit, by Qiagen (1 mentions)
Endotoxin removal beads, by Miltenyi Biotec (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
El800 plate reader, by Agilent Technologies (1 mentions)
Protein g antibody affinity chromatography, by GE Healthcare (1 mentions)
Cxcl4, by Merck Group (1 mentions)
11 protocols using chromogenic lal endotoxin assay kit
1
Characterization of LPS Interactions
2
mRNA Lipid Nanoparticle Formulation and Characterization
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The synthesized RBD-mRNA was encapsulated with lipid nanoparticles (LNPs) as described below (Tai et al., 2020 (link); Wang et al., 2022 (link)). Specifically, PNI Formulation Buffer with or without the synthesized mRNA was combined with GenVoy-ILM (lipid mixture) (Precision Nanosystems) at 3:1 ratio using NanoAssemblr Benchtop Instrument (Precision Nanosystems). The LNP-formulated mRNA was concentrated using 10 kDa Amicon Ultra-15 Centrifugal Filters (EMD Millipore) and stored at 4 °C until use. The endotoxin level (<1 EU/ml) of each LNP-encapsulated mRNA was measured using Chromogenic LAL Endotoxin Assay Kit (GenScript), and related particle size was measured using DynaPro NanoStar II Light Scattering Detector (WYATT Technology).
3
Purification and Endotoxin Removal of CPP Fusion Proteins
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The expression vectors were transformed into Rosetta 2(DE3) competent cells (Novagen, Darmstadt, Germany) for CPP fusion protein expression. The recombinant proteins were purified using a Ni2+-NTA Fast Start Kit from QIAGEN (Hilden, Germany, Cat. # 30600). A centrifugal filter unit from Millipore (Bedford, MA, USA, Cat. # UFC501008) was applied to remove the salt and to concentrate purified proteins. Protein quantitation was performed with a Pierce™ BCA protein assay kit from Thermo Fisher (Waltham, MA, USA, Cat. # 23227). Endotoxin was removed with endotoxin removal beads from Miltenyi Biotec (Germany, Cat. # 130-093-659). Endotoxin levels were determined by using a Chromogenic LAL Endotoxin Assay Kit from GenScript (Nanjing, China, Cat. # L00350).
4
Quantification of Serum LPS Levels
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Serum LPS was quantified in triplicate from mice chronically treated with water (n = 4) or LPS (n = 4) according to the manufacturer’s instructions (Chromogenic LAL Endotoxin Assay Kit, #L00350, Genscript) using an EL800 plate reader (Bio-Tek Instruments) with absorbance at 540 nm. Positive control wells with serum containing three increasing concentrations of LPS were also run in triplicate. The limit of detection for the kit is 0.01–1 EU/ml.
5
Preparation and Characterization of mAbs-LTF ICs
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Preparation and characterization of a mouse mAbs against huLTF (M860) in this laboratory have been reported in our previous work.35 (link) For preparation of LTF-IC, huLTF (2 µg/mL, Sigma-Aldrich) and M860 (2 µg/mL, mAbs of LTF), purified with protein G antibody affinity chromatography (GE Healthcare Life Sciences), or LTF-Abs were mixed in a sterile tube with gentle rotation at 37°C for an hour. ICs between LTF and M860 were separated from the uncoupled Ab and antigen using Sephadex Superfine G-75 column. The elutions of IC were pooled, desalted and concentrated. Endotoxin was removed by polymyxin B coupled beads repeatly and the level of endotoxin in IC was below 1 EU/mg which was detected by Chromogenic LAL Endotoxin Assay Kit (Genscript). ICs between ovalbumin (OVA) (Sigma-Aldrich) and M562 (mAbs of OVA) were used as control and prepared similarly.
6
Endotoxin Quantification in CXCL4 Stocks
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The concentration of endotoxin in CXCL4 stock solutions was measured by Chromogenic LAL Endotoxin Assay Kit (GenScript, cat. No: L00350C) according to the manufacturer’s instructions.
7
Endotoxin Contamination Testing of P(MPC-bl-(DMAEMA-co-BMA)) Polymers
8
Monocyte isolation and stimulation
9
Isolation and Stimulation of Human Monocytes
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Deidentified human buffy coats were purchased from the New York Blood Center following a protocol approved by the Hospital for Special Surgery Institutional Review Board. Peripheral blood mononuclear cells were isolated with Lymphoprep (Accurate Chemical) via density gradient centrifugation and monocytes were purified from peripheral blood mononuclear cells with anti-CD14 magnetic beads as recommended by the manufacturer (Miltenyi Biotec). 10 Monocytes were cultured overnight at 37 °C, 5% CO 2 in RPMI-1640 medium (Invitrogen) supplemented with 10% heat-inactivated defined fetal bovine serum (FBS) (HyClone; Fisher Scientific), penicillin-streptomycin (Invitrogen), L-glutamine (Invitrogen), and 20 ng/mL human macrophage colonystimulating factor (M-CSF). After at least 12-h culture, the cells were treated as described in the figure legends and were harvested and prepared for total RNA extraction, protein extraction, and flow cytometry. Cells were stimulated with CXCL4 (Sigma) and/or TLR8 ligand ORN8L (ChemGenes) as previously described 10 ; testing of various lots of CXCL4 using the Chromogenic LAL Endotoxin Assay Kit (Genscript) showed that CXCL4 preparations contributed 0.01 to 0.06 EU/mL final concentration of endotoxin in cultures, which does not contribute appreciably to synergy with ORN8L. 10
10
Chromogenic LAL Endotoxin Assay for Serum LPS
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A Chromogenic LAL Endotoxin Assay Kit (GenScript, Nanjing, China) was used to detect serum LPS. The protocol can be viewed on the website (https://www.genscript.com.cn/ , accessed on 23 Augest 2023).
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