Blood was sampled from the antecubital vein in two tubes: one 10 mL lithium-heparin-coated vacutainer for cell stimulation, and one 5,5 mL EDTA-coated vacutainer was analysed for complete blood cell count, red blood cells, leukocytes, and platelets using a Sysmex XN automated haematology analyser XN (Sysmex Corporation, Kobe, Japan). In brief, blood-samples from lithium-heparin tubes were diluted 1:1 in RPMI-1640 containing LPS, CML, A2E or DMSO as a mock control and incubated in a humidified 37 °C, 5% CO2 incubator for 4 h. LPS (from E.coli serotype O26:B6, Sigma-Aldrich, St. Louis, USA) was diluted to 5 mg/ml in DMSO and added to the blood-RPMI solution to obtain 100 ng/ml concentrations. CML (Carboxy-methyl Lysine modified Bovine Serum Albumin, PAB-26388-500, Nordic Biosite, Täby, Sweden) in DMSO and added to blood samples to a final concentration of 10 µg CML/ml, and A2E was added to blood cells as described above.
Lps from e coli serotype o26 b6
Manufactured by Merck Group
Sourced in United States
LPS from E. coli serotype O26:B6 is a laboratory reagent that is extracted from the cell walls of Escherichia coli bacteria. It contains lipopolysaccharide, a key component of the outer membrane of Gram-negative bacteria. This product can be used for research purposes in various applications.
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5 protocols using lps from e coli serotype o26 b6
1
Cytokine Release Assay for Inflammatory Markers
2
Inflammation and Phagocytosis Modulation
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Cells were either exposed to 0.1 μg/ml LPS from E. coli serotype O26:B6 (Sigma-Aldrich, Taufkirchen, Germany) for 24 hours to induce maximum stimulation, to 0.25 mM palmitic acid (PA, Sigma-Aldrich, Taufkirchen, Germany), to 20 μM fenofibrate (Tocris Bioscience, Bristol, United Kingdom) or to increasing concentrations of PEA (1, 3, 10, 30, 100, 300, 1,000 nM) for 30 minutes or 1 hour. PEA (molecular mass 299.5 Da) was obtained from Tocris Bioscience (Bristol, United Kingdom) and dissolved in 0.01% dimethyl sulfoxide (DMSO) according to the manufacturer’s instructions. We have previously shown that 30 minutes of PEA exposure was more effective at increasing bacterial phagocytosis of microglial cells than 24 hours of PEA stimulation [19 (link)]. A control group with DMEM containing 0.01% DMSO was included in all experiments. After stimulation, supernatants were stored at -20°C until measurement of cytokines and chemokines.
3
RAW 264.7 Macrophage Stimulation Assay
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Mouse macrophage cell line RAW 264.7 was cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin and 100 µg/mL streptomycin (DMEM-FBS) and maintained at 37°C in a 5% CO2 humidified incubator.
Cells were seeded in 35 mm-dishes or 24-well culture plates, lowed to adhere for 8 h at 37 °C in a humidified atmosphere of 5% CO2 prior to the addition of Lactobacillus reuteri supernatant (LrS) resuspended in medium (at a sub-cytotoxic dilution) and incubated for 4 h. Then, cultures were exposed to LPS from E. coli serotype O26: B6 (Sigma, St. Louis, MO, USA) at a final concentration of 15 µg/ml for 20 h. In all experiments, the cells were grown to 80–90% confluence and cell viability was assessed by the Trypan-blue assay (Sigma, St. Louis, MO, USA).
Cells were seeded in 35 mm-dishes or 24-well culture plates, lowed to adhere for 8 h at 37 °C in a humidified atmosphere of 5% CO2 prior to the addition of Lactobacillus reuteri supernatant (LrS) resuspended in medium (at a sub-cytotoxic dilution) and incubated for 4 h. Then, cultures were exposed to LPS from E. coli serotype O26: B6 (Sigma, St. Louis, MO, USA) at a final concentration of 15 µg/ml for 20 h. In all experiments, the cells were grown to 80–90% confluence and cell viability was assessed by the Trypan-blue assay (Sigma, St. Louis, MO, USA).
4
LPS-Induced Behavioral Changes in Mice
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LPS from E. coli serotype O26:B6 was obtained from Sigma Aldrich (St. Louis, MO, USA). The LPS was dissolved in sterile saline vehicle to a concentration of 0.1mg/ml. Mice were randomly assigned to treatment groups and received a single intraperitoneal injection of LPS (1.5mg/kg body weight) or an equivalent volume of sterile saline vehicle and returned to their home cage immediately following injection. The dose of LPS when administered during the peripubertal period has previously been demonstrated to decrease behavioral responses to estradiol in adulthood [8 (link), 10 (link), 18 (link), 19 , 32 (link)]. Consistent with previous reports [8 (link), 10 (link), 18 (link), 19 , 32 (link)], this dose of LPS produced moderate sickness behavior.
5
Bovine Mammary Immune Response to Pathogens
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On the day of the experiment, cows were randomly allocated to 5 treatment groups (group A, control, n = 4; group B, LPS, n = 4; group C, LPS+PRED, n = 4; group D, LTA, n = 4; and group E, LTA+PRED, n = 4), and mammary glands were challenged according to Figure 1 and as described elsewhere (Wall et al., 2016) . Briefly, immediately following morning milking, 2 quarters from each cow were injected via the teat canal and each quarter received a co-injection of 2 treatments from separate sterile syringes. The injections were performed by sterilizing each teat with gauze soaked in 70% ethanol and inserting a sterilized teat cannula. A 15-s massage in the cisternal direction was performed immediately after injection.
Treatments were prepared as follows: 0.2 μg of LPS (from E. coli serotype O26:B6, Sigma-Aldrich, St. Louis, MO) diluted in 10 mL 0.9% sterile saline; 20 μg of LTA (from S. aureus, Sigma-Aldrich) diluted in 10 mL of 0.9% sterile saline; 30 mg of PRED (prednisolone sodium phosphate, Santa Cruz Biotechnology, Dallas, TX) diluted in 10 mL of double distilled water; the control treatment was 10 mL of 0.9% sterile saline. Time of injection was designated as time/d 0. Each cow had 1 treatment and 1 control quarter (Figure 1).
Treatments were prepared as follows: 0.2 μg of LPS (from E. coli serotype O26:B6, Sigma-Aldrich, St. Louis, MO) diluted in 10 mL 0.9% sterile saline; 20 μg of LTA (from S. aureus, Sigma-Aldrich) diluted in 10 mL of 0.9% sterile saline; 30 mg of PRED (prednisolone sodium phosphate, Santa Cruz Biotechnology, Dallas, TX) diluted in 10 mL of double distilled water; the control treatment was 10 mL of 0.9% sterile saline. Time of injection was designated as time/d 0. Each cow had 1 treatment and 1 control quarter (Figure 1).
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