Bone marrow–derived DCs (BMDCs) were generated as described elsewhere (31 (link)). Briefly, bone marrow cells were harvested from femurs and tibias of female C57BL/6 mice, and single-cell suspensions were cultured at a density of 2 × 106 cells in Petri dishes containing 10 ml of complete RPMI 1640 supplemented with 200 U/ml (20 ng/ml) rmGM-CSF. An additional 5 ml of complete RPMI 1640 containing 20 ng/ml rmGM-CSF was added on days 3 and 5 after culture. Cells were collected from each dish and counted on day 6. BMDCs (1 × 106 cells per milliliter) in six-well plates were pulsed with stimuli for 3 h followed by 18 h of incubation with 10% FCS [note that StII is inactivated in the presence of serum (32 )]. Subsequently, BMDCs were analyzed by flow cytometry. Stimuli consisted of StII and LPS as positive control (both at 1 µg/ml), as well as StII and LPS mixed with pmxB (10 µg/ml) or StII that was purified previously using Detoxi-Gel Endotoxin Removing Gel column prepacked (Thermo Fisher Scientific, Waltham, MA). The StII purity was ∼99% and contained <20 EU/mg protein.
Detoxi gel endotoxin removing gel column
Manufactured by Thermo Fisher Scientific
Sourced in United States
Detoxi-Gel Endotoxin Removing Gel column is a lab equipment product designed to remove endotoxins from samples. It functions by binding and removing endotoxins from the sample solution.
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Lab products found in correlation
4 protocols using detoxi gel endotoxin removing gel column
1
Bone Marrow-Derived Dendritic Cell Stimulation
2
Purification of the Lectin ArtinM
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ArtinM was purified as previously described [2 (link), 3 (link)] from the saline extract of Artocarpus heterophyllus (jackfruit) seeds through affinity chromatography with immobilized carbohydrate columns. After purification, the ArtinM aliquots were passed over a Detoxi-Gel™ Endotoxin Removing Gel column (Thermo Scientific, Waltham, MA) to remove LPS and other endotoxins.
3
Isolation of Human Neutrophils
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All reagents were filtered through Detoxi-Gel Endotoxin Removing Gel Columns (Thermo Fisher Scientific) prior to neutrophil isolation. Human neutrophils were isolated using a density-gradient separation method (Ostrowski et al., 2020 (link)). Briefly, 30 ml of blood was collected in sterile BD Vacutainer EDTA (BD Biosciences, Mississauga, ON, Canada) tubes and layered on the top of PolymorphPrep (Progen, Wayne, PA, USA) in a 1:1 ratio. The layered mixture was spun at 500 g (acceleration 1 and deceleration 0) for 30 min to separate polymorphonuclear neutrophils (PMN) from mononuclear cells. PMN were washed with Hank’s balanced salt solution with calcium and magnesium (HBSS+/+; Wisent) and then subjected to red blood cell lysis using 0.2% NaCl followed by 1.6% NaCl for 30 s each. Neutrophils were resuspended in HBSS+/+ and used within 1 hr for all experiments.
4
Extraction and Purification of Charantin
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Extracts of the outer layer (the flesh, MCF) and of the seed part (the pulp, MCP) of MC were prepared according to a modification of a previously described method. 22 Briefly, the MCF and MCP (130 mg) were extracted using methanol at a 1 : 10 ratio. Homogenization was performed in a blender using 1-minute bursts at the highest speed for 12 minutes. The homogenized extract was filtered through cheesecloth. A rotary evaporator was used to remove most of the methanol, and any remaining methanol evaporated using a water bath at 100 °C. Charantin was provided by Xi'an Day Natural Technology (Xi'an City, China). Bacterial endotoxins were removed from the extracts and charantin using detoxi-gel endotoxin-removing gel columns (Thermo Scientific, Rockford, USA) and the endotoxins quantified were below the detection limit (<0.125 EU mL -1 ) as measured by the E-toxate kit based on the Limulus Amebocyte lysate assay.
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