Mice anesthetized with xylazine and ketamine were intra-rectally inoculated with the indicated dosage of ultrapure Escherichia coli LPS (InvivoGen) resuspended in 100 μL sterile saline as described12 beginning two days prior to C. albicans infection with repeated dosing every two days thereafter.
Ultrapure escherichia coli lps
Manufactured by InvivoGen
Sourced in Belgium
Ultrapure Escherichia coli LPS is a purified lipopolysaccharide extract from Escherichia coli. It is a key component of the outer membrane of Gram-negative bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Pam3csk4, by InvivoGen (2 mentions)
Ifn γ, by R&D Systems (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Fungizone, by Thermo Fisher Scientific (1 mentions)
Proline, by Thermo Fisher Scientific (1 mentions)
Tripure rna isolation reagent, by Roche (1 mentions)
Cytofix fixation buffer, by BD (1 mentions)
Endofit ovalbumin, by InvivoGen (1 mentions)
Cd4 t cell isolation kit, by STEMCELL (1 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (1 mentions)
Collagenase d, by Roche (1 mentions)
Mouse inflammation antibody array c1, by RayBiotech (1 mentions)
7 protocols using ultrapure escherichia coli lps
1
Intestinal LPS Priming for Candida Infection
2
Mycolactone Modulates LPS-Induced Inflammatory Response
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DRG cells were seeded at a density of 5 x 104 cells on coverslips previously inserted in the wells of 24 well-plates. Cells were exposed to 5 and up to 20 ng/ml mycolactone for 30 min prior to a 16 h stimulation with 1μg/ml LPS (Ultra pure Escherichia coli LPS, InvivoGen), in the presence of mycolactone. The release of CCL2, IL-6 and TNF-α into culture supernatant was assessed by ELISA using respectively the mouse CCL2 Uncoated ELISA kit (ThermoFisher scientific, 88-7391-22) and the mouse IL-6 and TNF-α ELISA MAX kits (Biolegend, 431301 and 430904). For IL-6 ELISA assay on SCs, SCs were seeded at 2 x 104 in 24 well plates coated with poly-L-Lysin. Twenty-four hours later, SCs were treated for 30 min with increasing doses of mycolactone, then stimulated by 1 μg/m LPS + 20 ng/ml IFN-γ (R&D Systems, 485-MI) for 16 h, in presence of mycolactone. Production of IL-6 and TNF-α by microglia was assessed in the culture supernatant of cells seeded at a density of 2.5 x 105 cells per well in 12 well-plates, treated overnight with mycolactone before being stimulated for 8 h with 100 ng/ml LPS, in the presence of mycolactone.
3
Murine Cell Culture Reagents
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Culture media, foetal bovine serum (FBS), horse serum (HS), penicillin-streptomycin, fungizone, proline, trypsin-EDTA and PCR primers were purchased from Invitrogen (Merelbeke, Belgium). Poly-L-lysine was obtained from Sigma (Bornem, Belgium) and ultrapure Escherichia coli LPS from InvivoGen (Toulouse, France). Percoll™/RediGrad™ reagent was acquired from GE Healthcare (Uppsala, Sweden). TriPure RNA Isolation Reagent was bought from Roche Diagnostic (Vilvoorde, Belgium) and iScript cDNA Synthesis Kit as well as IQ™ SYBR® Green supermix from BioRad Laboratories (Nazareth, Belgium). Culture plastic ware was purchased from Greiner Bio-one (Wemmel, Belgium).
4
Intestinal LPS Priming for Candida Infection
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Mice anesthetized with xylazine and ketamine were intra-rectally inoculated with the indicated dosage of ultrapure Escherichia coli LPS (InvivoGen) resuspended in 100 μL sterile saline as described12 beginning two days prior to C. albicans infection with repeated dosing every two days thereafter.
5
Preparation and Inactivation of Fungal Cell Stimuli
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The stimuli used were depleted zymosan, Pam3CSK4, ultrapure Escherichia coli LPS (all from Invivogen), or inactivated C. albicans ATCC 26555 yeasts obtained as previously reported (12 (link), 28 (link), 31 (link)). Briefly, yeasts were grown in YPD medium (1% yeast extract, 2% peptone, 2% glucose) at 28°C up to the late exponential growth phase (A600 of 0.6 to 1), collected, and washed with water. Cells were resuspended in water and maintained for 3 h at 28°C with shaking and afterwards at 4°C for 24 h (starved yeast cells). Starved yeast cells were inoculated (200 μg [dry weight] of cells per ml) in a minimal synthetic medium and incubated for 3 h at 28°C. For inactivation, yeast cells were resuspended (20 × 106 cells/ml) in BD Cytofix fixation buffer and incubated for 1 h at room temperature. After treatment, fungal cells were extensively washed in phosphate-buffered saline (PBS) and brought to the desired cell density in cell culture medium. Viable yeasts for in vitro assays were grown in YPD medium at 28°C up to the late exponential growth phase, collected, washed in PBS, and brought to the desired cell density in cell culture medium. All procedures were performed under conditions designed to minimize endotoxin contamination as described elsewhere (31 (link)).
6
Adoptive Transfer of OT-I and OT-II T Cells
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OTI (CD8+) and OT-II (CD4+) T cells were isolated from the spleen and LNs (axillary, brachial, inguinal, and popliteal) of OTI and OT-II mice using the appropriate EasySep CD8+ T cell (STEMCELL) or CD4+ T cell isolation kit (STEMCELL) according to the manufacturer’s protocol. The isolated OTI and OT-II cells were resuspended in saline, and 1x106 cells of each OTI and OT-II cells were injected into recipient C57BL/6 mice (female) via tail-vein injection (day 0).
On day 1 and day 7, the recipient mice were administered via hock subcutaneous injections with saline, 20 μg OVA as free OVA (EndoFit Ovalbumin, InvivoGen) or OVA-p(Man) (synthesized in house using the similar conjugation strategy as BLG-p(Man)). On day 16, mice were challenged with 5.0 μg OVA and 25 ng of ultrapure Escherichia coli LPS (InvivoGen) in 25 μL saline into each of the four hocks. On day 21, mice were sacrificed, and the spleen- and hock-draining LNs (dLNs) were collected. Spleens were mashed into a single cell suspension through 70 μm cell strainers, and lysed with ACK lysis buffer (Gibco). LNs were digested with 1 mg/mL Ca2+ supplemented Collagenase D (Roche) for 30 min at 37°C and mashed into a single cell suspension.
On day 1 and day 7, the recipient mice were administered via hock subcutaneous injections with saline, 20 μg OVA as free OVA (EndoFit Ovalbumin, InvivoGen) or OVA-p(Man) (synthesized in house using the similar conjugation strategy as BLG-p(Man)). On day 16, mice were challenged with 5.0 μg OVA and 25 ng of ultrapure Escherichia coli LPS (InvivoGen) in 25 μL saline into each of the four hocks. On day 21, mice were sacrificed, and the spleen- and hock-draining LNs (dLNs) were collected. Spleens were mashed into a single cell suspension through 70 μm cell strainers, and lysed with ACK lysis buffer (Gibco). LNs were digested with 1 mg/mL Ca2+ supplemented Collagenase D (Roche) for 30 min at 37°C and mashed into a single cell suspension.
7
Macrophage and Splenocyte Cytokine Assay
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M-CSF macrophages were plated in 96-well plates at a density of 50,000 cells in 200 μl of complete cell culture medium. Splenocytes were plated in 24-well plates at a density of 2.5 × 106 cells in 0.5 ml of complete cell culture medium. Cells were challenged with the indicated stimuli for 24 h and cell-free supernatants were then harvested and tested for TNF-α release using a commercial ELISA kit (eBioscience, San Diego, CA). The stimuli used were Pam3CSK4 (100 ng/ml), Ultrapure Escherichia coli LPS (100 ng/ml) (Invivogen, San Diego, CA), and inactivated yeasts of C. albicans (2 x 106 yeasts/ml). Triplicate samples were analyzed in each assay.
TNF-α and IL-6 levels in the secretomes produced by HSPCs were measured using commercial ELISA kits (eBioscience, San Diego, CA). Moreover, levels of 40 cytokines were determined in the secretomes using a mouse cytokine array (RayBio Mouse inflammation antibody array C1) according to the manufacturer's instructions (RayBiotech, Norcross, GA).
TNF-α and IL-6 levels in the secretomes produced by HSPCs were measured using commercial ELISA kits (eBioscience, San Diego, CA). Moreover, levels of 40 cytokines were determined in the secretomes using a mouse cytokine array (RayBio Mouse inflammation antibody array C1) according to the manufacturer's instructions (RayBiotech, Norcross, GA).
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