Bone marrow was isolated by flushing sterilized mouse femurs with 10 ml of RPMI medium. Bone marrow cells were cultured in tissue-culture-treated 6-well plates (Corning), in 4ml RPMI medium containing 10% FCS, 1% penicillin/streptomycin and 25 ng/ml murine GM-CSF (PeproTech), at a density of 106 cells per ml. On day 2, half of the medium was replaced with fresh medium containing 25 ng/ml murine GM-CSF. On day 3, the complete medium (containing non-adherent cells) was replaced with fresh medium containing 25 ng/ml murine GM-CSF. Non-adherent cells were harvested on day 6 by gentle washing with sterile endotoxin-free PBS (Sigma) and incubated with biotinylated anti-mouse CD11c (clone N418; BioLegend). CD11c+ cells (BMDCs) were purified by magnetic separation using streptavidin-coated magnetic beads (IMag Cell Separation System; BD). Isolated BMDCs were pulsed with ovalbumin (Grade V, Sigma, 100 μg/ml) for 16 hours. For some experiments, recombinant LCN2 (BioLegend) was added during the pulsing step at indicated concentrations. Ovalbumin-pulsed BMDCs were used for antigen presentation assays as described below.
Endotoxin free pbs
Manufactured by Merck Group
Sourced in United Kingdom, United States
Endotoxin-free PBS is a phosphate-buffered saline solution that has been processed to remove endotoxins. It is a commonly used buffer in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Lps from e coli o26 b6, by Merck Group (2 mentions)
Rmear11, by R&D Systems (2 mentions)
Ultrapure lps eb from escherichia coli 0111 b4, by InvivoGen (2 mentions)
Rmil 33, by Thermo Fisher Scientific (2 mentions)
Cxcl1, by R&D Systems (2 mentions)
Rmil 25, by Johnson & Johnson (2 mentions)
6 well tissue culture treated plates, by Corning (1 mentions)
Imag cell separation system, by BD (1 mentions)
Murine gm csf, by Thermo Fisher Scientific (1 mentions)
Tak 242, by Merck Group (1 mentions)
B6.129s4 ccr2tm1ifc j, by Jackson ImmunoResearch (1 mentions)
Control igg, by BioXCell (1 mentions)
Control rat igg, by BioXCell (1 mentions)
C57bl 6j, by Jackson ImmunoResearch (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Ova grade 3, by Merck Group (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
Endotoxin free pbs, by Lonza (1 mentions)
Ova grade 5, by Merck Group (1 mentions)
Endofree plasmid maxi kit, by Qiagen (1 mentions)
19 protocols using endotoxin free pbs
1
Isolation and Activation of Mouse Bone Marrow-Derived Dendritic Cells
2
HBV Envelope Protein Expression Plasmid
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pCMV-S2.S19 (link) encodes the small (S) and middle (preS2 + S) proteins of the HBV envelope (ayw subtype) under the control of the cytomegalovirus (CMV) immediate early gene promoter. The pMAS core plasmid (a kind gift of HL Davis) was used as the DNA encoding for the HBV capsid. The plasmid DNA used for immunization was purified using anion exchange chromatography columns (Plasmid Factory, Bielefeld, Germany), dissolved in endotoxin-free PBS (Sigma, St Louis, MO) at 1 mg/ml and stored at or below -20°C until use.
3
Neonatal LPS-induced Lung Inflammation
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Neonatal C57BL/6J and CCR2−/− (B6.129S4-Ccr2tm1Ifc/J) mice from Jackson Laboratories (Bar Harbor, ME) were inoculated with 3 µg/10 µl of LPS from E. coli O26:B6 (Sigma-Aldrich, St. Louis, MO), or endotoxin-free PBS (Sigma-Aldrich), intranasally on days of life 3, 5, 7, and 10. Selected mice were injected intraperitoneally with 10 mg/kg of the TLR4 inhibitor TAK-242 (MilliporeSigma, Burlington, MA) or dimethyl sulfoxide control (Sigma-Aldrich) dissolved in 40 µl of endotoxin-free PBS per mouse.
4
BALB/c Mice Infected with S. aureus
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Female BALB/c mice were purchased from Harlan (UK) and maintained at the University of Sheffield using standard husbandry procedures. The 7–8 week old mice were inoculated in the tail vein with 100 µl of S. aureus suspension in endotoxin-free PBS (Sigma) corresponding to 1×107 CFU per mouse. Viable bacteria in the inoculum were plated on BHI (plus appropriate antibiotics) after serial decimal dilution to confirm the accuracy of the bacterial dose. Mice were monitored and sacrificed at various time-points according to experimental design.
5
Production of Mouse Monoclonal Antibody 6B4
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Example 1
Production of the Mouse Monoclonal Antibody 6B4
Female BALB/c mice (4 wk old) were immunized by i.p. injections with 30 μg of purified recombinant S100A9 in 50 μl of endotoxin-free PBS (Sigma-Aldrich) mixed in an equal volume of complete Freund's adjuvant. Antibody responses were enhanced by injections 14 days later with S100A9 using incomplete Freund's adjuvant, and final boost was given on day 28 with proteins alone. On day 31, spleen cells from the immunized mice were fused with SP2 murine myeloma cells and cultured in hypoxanthine/amethopterin/thymidine selection medium.
Culture supernatants of the hybridomas were screened by ELISA using plates coated with 1 μg/ml recombinant proteins in 0.1 M carbonate buffer (pH 9.6). Positive hybridoma cells were cloned by limiting dilution. The mAb clone 6B4 showed the most distinctive recognition of the recombinant protein S100A9 and was isotyped as IgG1 kappa. Specificity of the 6B4 mAb was confirmed by ELISA and Western blot analysis (
6
Neonatal Lung Inflammation Model
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Neonatal C57BL/6J (wild type), Klrk1-/- (B6.Cg-Klrk1tm1Dhr/J, Nkg2d-/-) and Tcrd-/- (B6.129P2-Tcrdtm1Mom/J) mice were purchased from Jackson Laboratories (Bar Harbor, ME). Wild type and knock out mice were inoculated with 3μg/10μl of LPS from E. coli O26:B6 (Sigma-Aldrich, St. Louis, MO), or endotoxin-free PBS (Sigma-Aldrich), intranasally on day of life (DOL) 3, 5, 7, and 10 as described (28 (link)). Selected mice were injected intraperitoneally with 0.5 mg/kg of anti-IL17a antibody (clone TC11-18H10, BioLegend, San Diego, CA) or an IgG control (Rat IgG control, BioXCell, Lebanon, NH) on DOL 3, 4, 5, 6, 7, 10 and 12. Some mice were injected intraperitoneally with anti-NKG2D antibody (Clone HMG2D) or IgG control (10mg/kg) (BioXCell) 1 hr prior to each LPS treatment.
7
Preparation of Liver Fluke Homogenate and Excretory-Secretory Products
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Liver fluke homogenate (LFH) and ES products were prepared according to the protocol described by Smith et al33 (link) Briefly, for LFH, adult F. hepatica parasites were collected from bovine livers at an abattoir and placed in ice‐cold endotoxin‐free PBS (Sigma‐Aldrich). Within 3 hours of collection, all samples were rinsed and frozen at − 80°C. Subsequently, the frozen flukes were homogenized using a pestle and mortar. For ES, F. hepatica specimens were collected as described above. On return to the laboratory, all dead and damaged parasites were removed. Those remaining were rinsed and incubated at a density of 10 specimens per 5 mL of warm culture medium consisting of Roswell Park Memorial Institute‐1640 (RPMI‐1640; Invitrogen) supplemented with 25 mmol/L HEPES, 2% glucose and 25 μg/mL gentamycin in T‐25 tissue culture flasks for 4‐8 hours at 37°C and 5% CO2. The resulting supernatant was centrifuged at 600 g for 20 minutes at 4°C to remove debris. Finally, the centrifuged ES fraction was concentrated using 3 kDa centrifugal concentrators (Millipore). The resulting antigen suspensions were filtered through a 70‐μm strainer and then filter‐sterilized. Endotoxin was removed using Triton X‐114 phase separation as described by Flynn et al2 (link)
8
OVA-Induced Allergic Airway Inflammation Model
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Mice were sensitized to OVA (grade V) (Sigma, St. Louis, MO) by intraperitoneal (i.p.) injection of 100 μg OVA adsorbed on 1 mg alum (Sigma) in 0.5 mL endotoxin‐free PBS (Lonza, Basel, Switzerland) on days 0 and 7. Starting from day 21, mice were challenged for 30 min with an aerosolized solution of 1% OVA (grade III) (Sigma) in endotoxin‐free PBS for 2–8 consecutive days. Mice that were sensitized with OVA/alum and challenged with PBS served as controls.
9
Investigating Cytokine and Allergen Effects
10
Heterologous Prime-Boost Vaccine Regimes in Mice
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All animal experiments and procedures were performed according to the UK Animals (Scientific Procedures) Act Project Licence (PPL 30/2414 and 30/2889) and approved by the Oxford University Local Ethical Review Body. Age-matched female BALB/c mice (Harlan, UK), housed in specific-pathogen free environments, were vaccinated with equal amount of vaccines into each legs via the intramuscular route (i.m.) using either a heterologous prime-boost viral-vectored regime or protein-in-adjuvant prime-boost regime. For the viral-vector vaccinations, mice were primed with 1 × 108 i.u. ChAd63 and boosted 8 weeks later with 1 × 107 pfu MVA. Control vaccinations were performed with ChAd63 and MVA expressing green fluorescent protein (GFP). Vaccines were prepared in sterile endotoxin-free PBS (Sigma-Aldrich, UK). For the protein-in-adjuvant immunizations, the proteins were formulated with the adjuvant prior to vaccination as follows: Alhydrogel (85 μg Alhydrogel per dose was mixed with antigen in 20mM Tris buffer and incubated at room temperature (RT) for 1 h before injection); Matrix M (12 μg of Matrix M per dose in mice mixed with antigen in PBS); MF59 (mixed with antigen in PBS at a 1:1 volume ratio and 25 μL of MF59 per dose in mice); LMQ (30 μg of LMQ per dose in mice mixed with antigen in PBS).
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