Hippocampal microglia were isolated using a Percoll density gradient as previously described (Frank et al., 2006 (link)). We have previously shown (Frank et al., 2006 (link)) that this microglia isolation procedure yields highly pure microglia (Iba-1+/Cd163-/Gfap- mRNA). In the present experiments, immunophenotype and purity of microglia were assessed using real time RT-PCR. Microglia were suspended in DMEM+10% FBS and microglia concentration was determined by trypan blue exclusion. Microglia concentration was adjusted to a density of 1 x 104 cells/100 μl and 100 μl added to individual wells of a 96-well v-bottom plate. LPS (E. coli serotype O111:B4; Sigma-Aldrich; cat. no. L3012) was utilized to challenge microglia ex vivo as we have previously determined the optimal in vitro conditions under which LPS stimulates a microglial proinflammatory cytokine response (Frank et al., 2006 (link)). Cells were incubated with LPS (1, 10, and 100 ng/ml) or media alone for 2 h at 37 °C, 5% CO2. The plate was centrifuged at 1000 x g for 10 min at 4 °C to pellet cells and cells were then washed 1x in ice-cold PBS and centrifuged at 1000 x g for 10 min at 4 °C. Cell lysis/homogenization and cDNA synthesis were performed according to the manufacturer’s protocol using the SuperScript III CellsDirect cDNA Synthesis System (Invitrogen).
Lps e coli serotype o111 b4
Manufactured by Merck Group
Sourced in United States
LPS (E. coli serotype O111: B4) is a laboratory product produced by Merck Group. It is a lipopolysaccharide derived from the O111:B4 strain of Escherichia coli. The core function of this product is to serve as a research tool for studies involving endotoxin and innate immune responses.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 3 cellsdirect cdna synthesis system, by Thermo Fisher Scientific (1 mentions)
Control igg, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Biosera (1 mentions)
Stable glutamine, by Biosera (1 mentions)
Hank s balanced salt solution, by Biosera (1 mentions)
Rbc lysis buffer, by Thermo Fisher Scientific (1 mentions)
Garcinol, by Merck Group (1 mentions)
7 protocols using lps e coli serotype o111 b4
1
Isolation and Characterization of Hippocampal Microglia
2
LPS-Induced Sepsis in Chimeric Mice
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Eight weeks post-BMT, chimeras were injected i.p. with 5 mg/kg LPS (E.coli serotype O111:B4; Sigma–Aldrich MO, USA). Immediately after LPS injection, mice were placed on warming pads in order to prevent hypothermia and reduce mortality106 (link),107 (link). Mice were monitored daily for the next 7 days. All experimental procedures were performed under pathogen-free conditions. In cytokine neutralization experiments, 2 h post-LPS mice received either anti-IL-6 monoclonal neutralizing antibodies (2.5 mg/kg body weight, i.v.; Thermo Fisher, USA) or control IgG (Thermo Fisher, USA) in 200 μl of sterile saline via injection into the lateral tail vein.
3
Preparation of HMGB1 Proteins
4
Examining Fish Immune Response
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Following the randomized separation of six fish for the control group, the rest of the fish were divided into three equal groups: the LPS (E. coli, serotype O111: B4, Sigma-Aldrich Chemie, Deisenhofen, Germany, 2 mg/kg, IP) group, the LPS (2 mg/kg IP) + florfenicol (Nuflor flk, Intervet Ilac, Istanbul, 40 mg/kg, IM) group, and the florfenicol (40 mg/kg, IM) group, respectively. Following these applications, the fish were anesthetized with quinaldine (25 mg/L) for anesthesia and blood samples were taken from their tails at 1.5, 3, 6, 10, and at 24 hours.
5
Preparation of LPS and Garcinia Extracts
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LPS (E. coli serotype O111: B4, Sigma Aldrich, USA) was dissolved in 1X PBS to obtain a stock solution of 1mg/ml. Aliquots of the LPS stock were kept at -20 °C until the experiment. Similarly, stock solution of Garcinia methanolic extract (GME) was prepared by dissolving 10mg GME in 1ml of miliQ water. Again, the 20mM stock solution of Garcinol was prepared by dissolving Garcinol in 0.25% DMSO in miliQ water (Parasramk et al., 2012) . The stock solutions were then filter sterilized using 0.22µm syringe filters (HiMedia). 1µg/ml LPS was used to stimulate the cell lines (Kalita et al., 2022) .
6
Isolation and Stimulation of Resident Peritoneal Cells
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Resident peritoneal cells (RPCs) were harvested by washing the peritoneal cavities of naïve C57BL/6 mice with 10 mL of ice-cold Hanks’ balanced salt solution (Biosera, Nuaillé, France). Following the removal of red blood cells by the addition of 1x RBC lysis buffer (Invitrogen, Waltham, MA, USA), RPCs were resuspended in RPMI 1640 medium with stable glutamine (Biosera) supplemented with 0.5% bovine serum albumin (BSA, Biosera) and seeded into a 24-well cell culture plate (3.5 × 105 cells in 300 μL of culture medium per well). After incubating the cells for 4 h at 37°C and 5% CO2, the culture medium was replaced with fresh medium, and Iripin-1 (2 μM) or the corresponding amount of control buffer were added to certain wells. Following 30 min incubation at 37°C and 5% CO2, RPCs were stimulated by the addition of 1 μg/mL lipopolysaccharide (LPS, E. coli serotype O111:B4, Sigma-Aldrich, St. Louis, MO, USA) or were left unstimulated. Cell-free supernatants were collected after culturing the cells for 4 h at 37°C and 5% CO2 in the presence or absence of LPS and were stored at – 80°C until use. The concentrations of selected cytokines and chemokines in the supernatants were measured by commercial sandwich enzyme-linked immunosorbent assay (ELISA) kits as described later in the text.
7
Preparation of LPS and Garcinia Extracts
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LPS (E. coli serotype O111: B4, Sigma-Aldrich, USA) stock solution of 1 mg/ml was prepared in 1X PBS and stored in aliquots at -20 °C until the experiment. The methanolic extract of commercially available Garcinia herbal supplement (Himalaya, Lot No.: 11702264) was prepared for in vitro treatment. (-)-Hydroxycitric acid lactone (Sigma-Aldrich, USA, CAS No. 27750-13-6) stock solution of 5 mg/ml was prepared in miliQ water, and further dilution was done in DMEM. Similarly, Garcinol (Sigma-Aldrich, USA, CAS No.78824-30-3) stock solution was prepared by dissolving in 0.25%DMSO in miliQ water to make 20-mM stock solution, and further dilution was done in DMEM [36] (link).
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