C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA; stock no. 000664). Upon arrival, mice were housed on a 12 hour on/12 hour off light cycle in biohazard level 2 conditions, and acclimatized for at least two weeks to equilibrate their microbiota. A ketamine (85 mg/kg body weight; Ketasthesia, Henry Schein Animal Health, Dublin, OH, USA) and xylazine (14 mg/kg body weight; AnaSed; Akorn Inc., Decatur, IL, USA) cocktail was used to sedate the mice. Sample size was determined based on our previous gene expression study.50 (link) Four groups of 8- to 10-week-old mice with three mice in each group were used in these experiments (Fig. 1 A). Groups 1 and 2 were infected with 100 CFU WT B. thuringiensis/0.5 µL BHI, and Group 3 was infected with 100 CFU ΔslpA B. thuringiensis/0.5 µl BHI, as previously described.33 (link),34 At four hours postinfection, Group 2 was intravitreally treated with the synthetic TLR2/4 inhibitor OxPAPC (Invivogen, Carlsbad, CA, USA; 30 ng/µL) (WT+OxPAPC).34 Uninfected mice (Group 4) were used as control.
Oxpapc
Manufactured by InvivoGen
Sourced in United States
OxPAPC is a laboratory reagent used for cell culture applications. It is a synthetic phospholipid that is oxidized for use in experiments involving oxidative stress response and signaling pathways. The product specification and quality are maintained by InvivoGen to ensure consistent performance in research settings.
Automatically generated - may contain errors
Lab products found in correlation
Cli 095, by InvivoGen (9 mentions)
Proteinase k, by Thermo Fisher Scientific (4 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Tak 242, by Merck Group (2 mentions)
Celastrol, by InvivoGen (2 mentions)
Pam3csk4, by InvivoGen (2 mentions)
Bay11 7082, by Merck Group (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
48 well plate, by Corning (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Streptomyces chromofuscus, by Enzo Life Sciences (1 mentions)
1 2 dipalmitoyl sn glycero 3 phosphoethanolamine dppe, by Avanti Polar Lipids (1 mentions)
L α phosphatidylcholine egg pc, by Avanti Polar Lipids (1 mentions)
Pepinh myd, by InvivoGen (1 mentions)
Pd98059, by Selleck Chemicals (1 mentions)
Sb203580, by Selleck Chemicals (1 mentions)
Sp600125, by AG Scientific (1 mentions)
C57bl 6j mice, by Jackson ImmunoResearch (1 mentions)
Anased, by Akorn (1 mentions)
Ketasthesia, by Henry Schein (1 mentions)
28 protocols using oxpapc
1
Murine Model of Bacillus thuringiensis Infection
2
Murine Model of Bacillus thuringiensis Endophthalmitis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
B. thuringiensis subsp. Galleriae NRRL 4045 (WT) or its isogenic S-layer protein-deficient mutant (∆slpA) was used to initiate experimental endophthalmitis in mice, as previously described 33,34 . WT B. thuringiensis and ΔslpA B. thuringiensis were grown to early stationary phase in brain heart infusion (BHI; VWR, Radnor PA) broth for 18 hours and diluted to 100 CFU/0.5 µl for injection into the mid-vitreous. Four groups of C57BL/6J mice with three mice in each group were used in these experiments (Figure 1A). The first two groups were infected with 100 CFU WT B. thuringiensis/0.5 µl BHI, and the third group was infected with 100 CFU ΔslpA B. thuringiensis/0.5 µl BHI,as previously described 33, 34 . At 4 hours postinfection, the second group of WT B. thuringiensis-infected mice was intravitreally treated with the synthetic TLR2/4 inhibitor OxPAPC (Invivogen; 30 ng/μl) (WT+OxPAPC) 34 . Uninfected mice were used as control .
3
Intestinal Epithelium Inflammation Modulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HT-29 and T84 cells were grown in 48-well plates (Corning). For agonist experiments, epithelial cells were incubated for 3 h with AAF/II fimbrial extract or 100 ng/mL TLR2 and TLR4 agonists BLP (InvivoGen) and LPS (E. coli O55:B5, Sigma), respectively. Then, the cells were washed and incubated for 3 h at 37°C under 5% CO2. Supernatants were collected for measuring Interleukin-8 (IL-8) levels. For EAEC infections, cells were incubated for 30 min, respectively with McCoy's or DMEM- F12 medium in the presence or absence of TLR2 and TLR4 antagonist OxPAPC (InvivoGen) or TLR4 antagonist TAK-242 (Merck), as instructed by the manufacturer. The cells were washed and incubated in medium supplemented with gentamicin (50 μg/mL) at 37°C under 5% CO2 for 3 h. Supernatants were then collected to measure inflammatory biomarkers.
4
TLR2 Binding Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
1,2-Dipalmitoyl-sn-glycero-3-phospho-ethanolamine
(DPPE) and egg PC (l -α-phosphatidylcholine) were purchased
from Avanti Polar Lipids (Alabaster, AL). Recombinant mouse TLR2-Fc
protein was purchased from R&D Systems (Minneapolis, MN). Phospholipase
D (PLD) from Streptomyces sp. and Streptomyces chromofuscus were obtained from Enzo
Life Sciences (Farmingdale, NY). Horseradish peroxidase-labeled goat
anti-human IgG Fc antibody was purchased from Millipore (Billerica,
MA). ABTS solution substrate for horseradish peroxidase was from Invitrogen
(Grand Island, NY). OxPAPC was from InvivoGen (San Diego, CA). The
fluorenemethyl ester of 3,6-dioxohexanoic acid (DOHA-Fm), CEP-modified
human serum albumin (CEP-HSA) and chicken egg ovalbumin (CEP-CEO),
and polyclonal rabbit anti-CEP-KLH antibody were prepared as described
previously.1 (link) Mouse anti-human TLR2 LEAF
antibody (CD282) was from BioLegend (San Diego, CA). Calcein AM and
Accutase were from BD Biosciences (San Jose, CA). Goat anti-mouse
IgG-AlexaFluor 488 was from Invitrogen (Carlsbad, CA). All other chemicals
were from Sigma-Aldrich and were analytical grade.
(DPPE) and egg PC (
from Avanti Polar Lipids (Alabaster, AL). Recombinant mouse TLR2-Fc
protein was purchased from R&D Systems (Minneapolis, MN). Phospholipase
D (PLD) from Streptomyces sp. and Streptomyces chromofuscus were obtained from Enzo
Life Sciences (Farmingdale, NY). Horseradish peroxidase-labeled goat
anti-human IgG Fc antibody was purchased from Millipore (Billerica,
MA). ABTS solution substrate for horseradish peroxidase was from Invitrogen
(Grand Island, NY). OxPAPC was from InvivoGen (San Diego, CA). The
fluorenemethyl ester of 3,6-dioxohexanoic acid (DOHA-Fm), CEP-modified
human serum albumin (CEP-HSA) and chicken egg ovalbumin (CEP-CEO),
and polyclonal rabbit anti-CEP-KLH antibody were prepared as described
previously.1 (link) Mouse anti-human TLR2 LEAF
antibody (CD282) was from BioLegend (San Diego, CA). Calcein AM and
Accutase were from BD Biosciences (San Jose, CA). Goat anti-mouse
IgG-AlexaFluor 488 was from Invitrogen (Carlsbad, CA). All other chemicals
were from Sigma-Aldrich and were analytical grade.
5
Protease and Inhibitor Procurement
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteinase K was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Bay11-7082 and SP600125 were purchased from A.G. Scientific (San Diego, CA, USA). PD98059 and SB203580 were purchased from Selleck (Houston, TX, USA). Pepinh-MYD, OxPAPC, and CLI-095 were purchased from Invivogen (San Diego, CA, USA).
6
Inhibition of TLR1/2 and TLR2/6 in Myeloid DCs
7
Modulation of Tracheal Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tracheal epithelial cells were incubated with either 3 µg/mL Rlow LAMPs or 5 µg/mL Rhigh LAMPs in the presence or absence of signaling inhibitors. TLR-2/4 signaling inhibitor OxPAPC (oxidated 1-palmitoyl-2-arachidonyl-sn- glycero-3-phosphorylcholine), TLR-4 inhibitor CLI-095, and NF-κB inhibitor Celastrol were purchased from Invivogen (Invivogen; San Diego, CA). Cells were co-incubated with either 10 µg/mL or 30 µg/mL OxPAPC and LAMPs for 6 hours, in accordance with the manufacturer's instruction. Cells were pre-incubated with 1 µg/mL CLI-095 for 6 hours before exposure to LAMPs and then further incubated for 6 hours after LAMP exposure. Pre-incubation of cells with 5 µM Celastrol for 30 minutes was done before LAMP exposure for 6 hours. All experiments were performed with 6 replicates each.
8
Isolation and Culture of Human Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dulbecco’s modified Eagle’s medium (DMEM), α-Minimum Essential Medium (α-MEM), antibiotic-antimycotic (anti-anti), fetal bovine serum (FBS), and phosphate-buffered saline (PBS) were purchased from the Life Technologies (Grand Island, NY). OxPAPC, CLI-095, and monophosphoryl lipid A (MPLA) were purchased from the Invivogen (San Diego, CA). Fibroblast growth factor-2 (FGF-2) was obtained from the R&D System (Minneapolis, MN). Tryptic soy broth was purchased from the Acumedia (Lansing, MI). Agar was obtained from the Fisher Scientific (Pittsburgh, PA). Human LL-37 ELISA kit was purchased from the Hycult Biotech (Plymouth Meeting, PA). LL-37 peptide was purchased from the AnaSpec (Fremont, CA). GW0742, trypsin IIS, and ampicillin (Amp) were purchased from the Sigma-Aldrich (St. Louis, MO). Collagenase type I was purchased from the Worthington (Lakewood, NJ). 1,25-dihydroxy vitamin D3 was purchased from the Cayman Chemical (Ann Arbor, MI).
9
Optimizing rhHSP27 for Endotoxin-Free Use
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To avoid endotoxin contamination of rhHSP27, cells were preincubated with polymyxin B (PMB, 10 μM; 30-60 min) and rhHSP27 solutions were also treated with PMB prior to their use. To block TLR3, HMEC were pre-incubated with the neutralizing anti-hTLR3 (antiTLR3 mAb; 20 μg/ml) for 1 h (eBioscience, San Diego, CA, USA). To block the signaling of TLR2 and TLR4 induced by LPS, OxPAPC (30 μg/ml) was added (InvivoGen). To block CXCR2, HMEC were pretreated with 200 nM SB225002 (CXCR2 antagonist) for 30 min or with 10 μg/ml of neutralizing anti-CXCR-2/IL-8 RD (Clone 48311) for 1 h (R&D Systems). For collection of conditioned media (CM), confluent cells were grown overnight in FCS-free DMEM then fresh medium (4 ml/T-75 flask) was added for 6 h before to be collected.
10
Inhibition of TLR2/4 Signaling Impacts Mineralization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TLR2/4 signaling was inhibited with OxPAPC (InvivoGen, San Diego, CA, USA), a mixture of oxidized phospholipids competing upstream with the TLR2/4 accessory proteins. Additionally, in a separate set of experiments, a pivotal downstream mediator of TLR2/4 signaling, TGF-β-associated kinase – TAK1, was also inhibited with 5Z-7-oxozeaenol (Sigma-Aldrich). Human SCAPs (passage 3) were seeded onto 48-well plates at a cell density of 2 × 104 cells/well and incubated with culture medium. Upon reaching confluency, the spent media were decanted and 0.5 mL from the different mineralizing conditioned media containing either 5 μM OxPAPC (reconstituted in chloroform) or 6 μM 5Z-7-oxozeaenol (reconstituted in DMSO) were added. The cells were cultured for 14 days, with the mineralizing conditioned media and the inhibitors being refreshed every 3 days. Cells incubated with the different mineralizing conditioned media containing chloroform and DMSO, served as controls. Following, an in vitro functional mineralization assay based on AR-S staining and quantification was performed as described above.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!