Escherichia coli 026 b6

Purified naïve splenic B cells were isolated from mice by first using a percoll (GE Healthcare, IL, USA) gradient (80/65/50% in PBS, cells collected from 80/65% interface), followed by negative isolation with a B cell isolation kit (Miltenyi Biotec), as described previously [9 (link), 12 (link), 40 (link)]. Cells were labeled with division tracking dye, Cell Trace Violet (CTV). Purity of B cell population was verified as >95% B220⁺ CD19⁺ by flow cytometry. Labeled B cells were stimulated with lipopolysaccharide (LPS) derived from Escherichia coli 026:B6 (15 µg/mL; Sigma). For in vitro differentiation studies, B cells were stained using antibodies to CD138 (clone 281-2, BD Pharmingen). Triplicate plates were set up on the first day and left undisturbed – one plate was analyzed at each timepoint. All lymphocytes were incubated at 37 °C with 5% CO₂ and humidity control.

For determination of whole-blood culture LPS-stimulated cytokine concentrations, uncentrifuged whole-blood samples collected in K2-EDTA tubes were diluted 1:9 with Roswell Park Memorial Institute 1640 medium (Sigma, UK) supplemented with 1% antibiotics, 1% l-glutamine, and 1% nonessential amino acids (BioScience, UK) (22 (link)). Subsequently, diluted blood samples were cultured in 12-well plates (Greiner Bio-one, UK) with 0.5 μg bacterial LPS/mL (Escherichia coli 026:B6; Sigma, UK), at a final concentration of 0.05 μg/mL. Cultures were incubated at 37°C for 24 h before centrifugation at 700 × g for 5 min at room temperature to isolate supernatant, which was stored at –20°C until analysis. A human cytokine premixed 5-Plex Panel (TNF-α, IL-6, IL-8, IL-1β, IL-10; R&D Systems Europe Ltd) was used to measure concentrations of cytokines in the whole-blood culture supernatant in a 1:2 dilution using a Luminex 200 with xPONENT software 3.1.
Measurement of monocyte count of each blood sample was performed by the Pathology Department at the Royal Berkshire Hospital (Reading, UK). Cytokine production was corrected for the number of monocytes in the whole-blood sample, expressed as mg × 10³ monocytes.

For the determination of whole blood culture LPS-stimulated cytokines, blood samples collected into K₂-EDTA tubes were not centrifuged and stored at 4°C until processing as previously described (24 ). Briefly, whole-blood samples were diluted 1:9 with Roswell Park Memorial Institute 1640 medium (Sigma) supplemented with 1% antibiotics, 1% l-glutamine, and 1% nonessential amino acids (Bioscience). Diluted blood samples were cultured in 12-well plates (Greiner Bio-one) with 0.5 μg bacterial LPS/mL (Escherichia coli 026:B6; Sigma), at a final concentration of 0.05 μg/mL. Whole blood cultures were subsequently incubated at 37°C for 24 h before centrifugation at 700 × g (1000 rpm) for 5 min at room temperature to isolate the supernatant, which was stored at −20°C until analysis. Measurement of the monocyte count of each sample was performed by the Pathology Department at the Royal Berkshire Hospital (Reading, UK). A human cytokine premixed 5-Plex Panel (TNF-α, IL-6, IL-8, IL-1β, IL-10; R&D Systems Europe Ltd.) and a Luminex 200 with xPONENT software 3.1 was used to measure the concentrations of cytokines in the whole blood culture supernatant in a 1:2 dilution. Cytokine production was corrected for the number of monocytes in the whole-blood sample (micrograms × 10³ monocytes).

Sodium dodecyl sulfate (SDS), sodium tetraborate, and sodium hydroxide were obtained from Merck (Darmstadt, Germany). Fluorescein isothiocyanate (FITC), (1-3)-β-D-glucans were purchased from Sigma - Aldrich (St. Louis, MO, USA). Lipopolysaccharides from Escherichia coli 0111:B4 (Lot # 024M4019V), Escherichia coli 055:B5(Lot #025M4040V), Escherichia coli 026:B6 (Lot # 053M4060V), Escherichia coli 0127:B8 (Lot # 103M4051V), Salmonella enterica serotype enteritidis (Lot # 064M4035V), Pseudomonas aeruginosa 10 (Lot # 100M4101V), Salmonella enterica serotype typhimurium (Lot # 093M4088V), Escherichia coli J5 (Rc mutant, rough strains) (Lot # 053M4112V), Salmonella typhosa (Lot # 063M4017V), Escherichia coli F583 (Rd mutant, rough strains) (Lot # 055M4005V), Salmonella enterica serotype minnesota (Lot # 064M4015V), Escherichia coli 0128:B12 (Lot # 063M4014V), Klebsiella pneumoniae (Lot # 111M4038V), Escherichia coli K-235 (Lot # 031M4076V), Salmonella enterica serotype abortusequi (Lot # 104M4064V), and Serratia marcescens (Lot # 013M4078V) were purchased from Sigma Aldrich (St. Louis, MO, USA). Buffers were prepared with ultrapure water obtained from Merck Millipore (Millipore, Bedford, MA, USA). All other reagents were of analytical grade. Experiments were carried out using freshly prepared and filtered solutions.

Adapted from Kizil et al., 2013 (link) study, cerebroventricular microinjection was performed on the adult zebrafish to induce neuroinflammation. Lipopolysaccharide (LPS) derived from Escherichia coli 026: B6 (Sigma Aldrich, St. Louis, United States ) was used as an inflammatory agent for the induction. Firstly, a small opening was incised on the zebrafish skull using a 30G needle on the cranial bone close to the midline located above the optic tectum. The slit exposes the zebrafish’s cerebroventricular fluid (CVF), allowing LPS solution to be microinjected using thin glass capillaries without damaging the brain. This injection can disperse the LPS solution throughout the brain, targeting the ventricular and periventricular cells, inducing generalized neuroinflammation. For this study, 100 nL of 2 mg/ml LPS were injected into zebrafish in groups 6-10, and distilled water of the same volume was injected into zebrafish in group 5.