Large amounts (1 × 1010) of non-labelled merozoites were collected and GPIs not linked to proteins were extracted with chloroform-methanol-water (10:10:3, by volume) by sonication, dried under a nitrogen stream and recovered in the n-butyl alcohol phase after partitioning between water-saturated n-butyl alcohol and water (1:1, by volume) by centrifugation. GPIs were precipitated under a stream of nitrogen to remove contaminating phospholipids [18 (link)]. GPIs were then separated by TLC on 0.5 mm silica gel 60 plate (Merck, GPIs from 5 × 109 parasites/plate) using a chloroform-methanol-water (4:4:1, by volume) solvent system, with spots of labelled GPIs used as tracers. TLC plates were scanned for radioactivity using a Berthold LB 2842 linear analyser and areas corresponding to individual GPIs were scraped off the plate, re-extracted with chloroform-methanol-water (10:10:3, by volume) by sonication (only half of the material is estimated to be recovered) and residual silica was removed by water-saturated n-butyl alcohol/water partition. GPIs were stored at −20 °C in n-butyl alcohol until use. Absence of endotoxin in each GPI was checked with the Pierce® Limulus Amebocyte Lysate Chromogenic Endotoxin Quantitation kit according to the manufacturer’s instructions (Thermo Scientific).
Pierce limulus amebocyte lysate chromogenic endotoxin quantitation kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce Limulus Amebocyte Lysate (LAL) Chromogenic Endotoxin Quantitation Kit is a laboratory reagent used to detect and quantify endotoxin levels in various samples. It utilizes the LAL assay, a sensitive and specific method for the detection of bacterial endotoxins.
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Lab products found in correlation
Poly 1 c, by InvivoGen (2 mentions)
Anti cd40 fgk4, by BioXCell (2 mentions)
Rpmi 1640 cell culture medium, by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
N acetylcysteine nac, by Merck Group (1 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
Polymyxin b pmb, by Merck Group (1 mentions)
Quanti blue, by InvivoGen (1 mentions)
Adenosine triphosphate (atp), by InvivoGen (1 mentions)
Antibodies against actin, by Merck Group (1 mentions)
Sp600125, by Cell Signaling Technology (1 mentions)
Pd98059, by Cell Signaling Technology (1 mentions)
Sb203580, by Cell Signaling Technology (1 mentions)
N formyl methionyl leucyl phenylalanine, by Merck Group (1 mentions)
Lps from escherichia coli o111 b4, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Cytochalasin b cytb, by Merck Group (1 mentions)
Direct detect infrared spectrometer, by Merck Group (1 mentions)
Pierce high capacity endotoxin removal spin column, by Thermo Fisher Scientific (1 mentions)
14 protocols using pierce limulus amebocyte lysate chromogenic endotoxin quantitation kit
1
Extraction and Purification of Plasmodium GPIs
2
Vaccinia and ova-psDNA Immunization
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6–8-week-old C57BL/6 (CD45.2) mice were immunized with 1E3 or 1E4 colony-forming units (CFU) of Vaccinia Western Reserve or 5 µg of poly I:C (Invivogen) with or without 5 µg of anti-CD40 (FGK4.5, BioXcell)and 10 µg of ova-psDNA or ova in 50 μL volume by footpad injection. Endotoxin levels were quantified using the Pierce Limulus Amebocyte Lysate Chromogenic Endotoxin Quantitation kit (Thermo Scientific) to be less than 0.5 EU/mg for either ova or ova conjugated to psDNA.
3
Vaccinia and CHIKV Infection Protocols
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6–8-week-old C57BL/6 (CD45.2) mice were immunized with 1E4 plaque-forming units (PFU) of Vaccinia Western Reserve or 5 μg of poly I:C (Invivogen) with or without 5 μg of anti-CD40 (FGK4.5, BioXcell) and 20 μg of ova-psDNA or ova in 50 μL volume per footpad injection. Endotoxin levels were quantified using the Pierce Limulus Amebocyte Lysate Chromogenic Endotoxin Quantitation kit (Thermo Scientific) to be less than 0.5 EU/mg for either ova or ova conjugated to psDNA. For CHIKV infections, 4 week old C57BL/6 mice were inoculated with 103 PFU of CHIKV 181/25 or CHIKV AF15561 (WT CHIKV) in a 10 μL volume in both rear footpads.
4
Buckwheat Polysaccharide Fractions Extraction
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Buckwheat (F. esculentum) was purchased from Changjingzhongye Inc., China. Buckwheat seed was used to extract BPFs as previously reported method, which uses 95% ethanol to precipitate polysaccharide fractions [11 ]. The total sugar content was 82.1% as indicated by the assays using phenol–sulfuric acid colorimetric method. The average molecular weights of BPFs were estimated to be 23,000 as measured by high-performance gel filtration chromatography. The components of BPFs mainly include glucose, galactose, fucose, arabinose, rhamnose,, and xylose. The endotoxin contamination of BPF was around 70.1 EU/mg as detected by a Pierce Limulus amebocyte lysate Chromogenic Endotoxin Quantitation Kit (Thermo Scientific, Rockford, IL, USA), which is considered to be insignificant for various bioactive products. BPFs dissolved in RPMI 1640 cell culture medium were applied to macrophages as stimuli. RPMI 1640 cell culture medium and LPS (serotype 0111:24; Sigma) was used as negative and positive controls, respectively.
5
Macrophage Inflammatory Pathway Inhibition
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Antibodies against proIL-1β, iNOS, COX-2, NF-κB SN50 cell permeable inhibitory peptide (sc-3060) and pyrrolidine dithiocarbamic acid ammonium salt (PDTC) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against actin, LPS, pharmaceutical inhibitors, N-acetyl cysteine (NAC), polymyxin B (PMB) and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). PD98059, SB203580, SP600125 and antibodies against phospho-proteins were obtained from Cell Signaling Technology (Beverly, MA, USA). The NF-κB reporter reagent (QUANTI-Blue) and ATP were obtained from InvivoGen (San Diego, CA, USA). ELISA kits were obtained from Affymetrix eBioscience (San Diego, CA, USA). The intracellular ROS indicator was obtained from Molecular Probes (Eugene, OR, USA). The florescent E. coli particles and Pierce Limulus Amebocyte Lysate (LAL) Chromogenic Endotoxin Quantitation Kit were obtained from Thermo Fisher Scientific (Waltham, MA, USA). M-CSF was obtained from Peprotech (London, UK). Lipid IVa was obtained from MyBioSource (San Diego, CA, USA).
6
Determination of Silk Endotoxin Levels
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The preferred kit to determine the endotoxin levels for these experiments was the Pierce Limulus Amebocyte Lysate (LAL) Chromogenic Endotoxin Quantitation Kit (Thermo Scientific Cat# 88282), which uses UV absorbance at 405–410 nm to determine endotoxin concentration. This kit has a working detection range of 0.1–1.0 EU/mL. Endotoxin levels above 1.0 EU/mL were extrapolated using an experimentally determined standard curve equation.
Because testing a solid piece of silk material interfered with the UV absorbance and confounded the endotoxin level readings, 1 mL of endotoxin-free water was added to samples after endotoxin destruction treatments. Sample/water mixtures were vigorously vortexed for >5 min to break apart the silk material and remove endotoxin from the silk and the container into the water; 50 µL of the water was then used for endotoxin testing.
Because testing a solid piece of silk material interfered with the UV absorbance and confounded the endotoxin level readings, 1 mL of endotoxin-free water was added to samples after endotoxin destruction treatments. Sample/water mixtures were vigorously vortexed for >5 min to break apart the silk material and remove endotoxin from the silk and the container into the water; 50 µL of the water was then used for endotoxin testing.
7
CBG Characterization and Endotoxin Assay
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CBG, with purity greater than 99%, was purchased from the Chinese National Institute for Food and Drug Control. It was dissolved in dimethyl sulfoxide (DMSO) from Sigma and stored in stock concentration of 20 mg/ml at -20°C. The Pierce Limulus Amebocyte Lysate (LAL) Chromogenic Endotoxin Quantitation Kit was purchased from Thermo Scientific. As a reference for mass spectrometry analysis showed in S1 Fig , CBG (C1272) was bought from Sigma-Aldrich, Sweden, dissolved to 1 mg/ml in MeOH. LPS from Escherichia coli O111:B4, adenosine 5′-triphosphate (ATP), staurosporine solution, cytochalasin B (CytB) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) were purchased from Sigma. The caspase-1 inhibitor Z-YVAD-FMK was from MBL. The lactate dehydrogenase (LDH) Cytotoxicity Detection Kit PLUS was from Roche Applied Science.
8
Serum Endotoxin Quantification in Mice
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Serum endotoxin concentration was measured using the Pierce Limulus Amebocyte Lysate (LAL) Chromogenic Endotoxin Quantitation Kit (Thermo Fisher Scientific, 88282). Blood samples were collected from WT and AOAH-deficient mice via incision of the tail vain. Samples were allowed to clot for 15 min at room temperature followed by centrifuging at 2,000 x g for 10 min at 4°C. Serum supernatant was then diluted 1:50 with endotoxin-free H2O and the assay was performed following the manufacturer’s instructions.
9
Purification of Mutant ADI Protein
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ADI (amino acids 1 to 411, D277A) previously cloned into the expression vector pET151/D-TOPO [13 (link)], was expressed in E. coli BL21 Star (DE3) cells and purified by immobilised metal ion affinity chromatography (IMAC) as previously reported [12 (link)]. Endotoxins were removed by washing the IMAC-bound protein with Triton X-114 [20 (link)]. Final protein concentration was determined using a Direct Detect infrared spectrometer (Millipore, Burlington, MA, USA) and endotoxin levels were measured using the Pierce Limulus amebocyte lysate (LAL) chromogenic endotoxin quantitation kit (Thermo Fisher Scientific, Waltham, MA, USA).
10
Quantifying Endotoxin in Phage Samples
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Purified phage preparations and bacterial debris were checked once for endotoxin content using a Pierce Limulus amebocyte lysate (LAL) Chromogenic Endotoxin Quantitation Kit (Thermo Fisher, Sweden) according to the manufacturer’s instructions.
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