Primary human keratinocytes were supplied by Lonza and cultured in Growth Medium 2 with supplement (C-20011, C-39011; PromoCell). Primary umbilical vein endothelial cells were supplied by Lifeline Cell Technology and cultured in endothelial cell growth medium (C-22010; PromoCell). Mouse primary keratinocytes were isolated from the tails of C57Bl/6 mice and cultured as previously [42 (link)]. Peripheral blood mononuclear cells (PBMCs) were isolated from donor blood by Ficol Histopaque 1077, then frozen at 5x106/mL in FBS/DMSO until use. Cells were thawed and cultured in RPMI + 10% hiFBS. T lymphocytes were depleted from PBMCs using positive selection for CD2 by MACS standard protocol (Militenyi). All cells were maintained at 37°C and 5% CO2. For PBMC-NHEK coculture, NEHKs were plated at 4x104/mL in keratinocyte growth media in TC-treated 96 well plates and grown for 48 h. The media was then aspirated and replaced with 100uL 1x106/mL PBMCs in RPMI + 10% FBS. For cell lysis, 0.05% triton X-100 (Sigma) was added 5 min. Other cell treatments are 5 μM caspase-1 inhibitor YVAD-fmk (R&D Systems), 50 μM SpeB inhibitor E-64 (Sigma), 20 μg/mL anti-IL-18 IgG (Abcam), or 10 μM caspase-1 inhibitor VX-765 (Invivogen), for the full duration of the experiment.
Vx 765
Manufactured by InvivoGen
Sourced in United Kingdom, United States
VX-765 is a laboratory equipment product manufactured by InvivoGen. It is a selective inhibitor of caspase-1, an enzyme that plays a key role in the inflammatory response. VX-765 can be utilized in research studies to investigate the involvement of caspase-1 in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Nigericin, by InvivoGen (7 mentions)
Nigericin, by Merck Group (5 mentions)
Adenosine triphosphate (atp), by Merck Group (5 mentions)
Mcc950, by Adipogen (4 mentions)
Nocodazole, by Merck Group (3 mentions)
Ucn 01, by Merck Group (3 mentions)
Glutamax medium, by Thermo Fisher Scientific (3 mentions)
Colchicine, by Merck Group (3 mentions)
Elisa, by R&D Systems (3 mentions)
Lipopolysaccharides (lps), by InvivoGen (3 mentions)
Fetal calf serum (fcs), by Lonza (3 mentions)
Calyculin a, by Merck Group (2 mentions)
Mcc950, by InvivoGen (2 mentions)
Z ietd fmk, by R&D Systems (2 mentions)
Mg132, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Opti mem media, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Yvad fmk, by R&D Systems (1 mentions)
Triton x 100, by Merck Group (1 mentions)
20 protocols using vx 765
1
Primary Immune Cell Coculture Protocol
2
Cytokine Quantification from Monocytes
3
Mycobacterial Infection of Macrophages
4
Prenatal Inflammasome Inhibition and Offspring Behavior
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pregnant C57BL/6 mice were given three intraperitoneal injections of either VX-765 (50 mg/kg/d, caspase-1 inhibitor, InvivoGen), MCC950 (50 mg/kg/d, NLRP3-inflammasome inhibitor, InvivoGen), or saline as vehicle. Drugs were injected once per day starting on embryonic day (E)12.5 until E14.5. Behavioral assays were performed on the male offspring when they were eight weeks old.
5
VX-765 Inhibits HIV Replication
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
VX-765 (Invivogen) was dissolved in 20% cremophor and injected intraperitoneally (i.p.) in HIV-1 infected humanized mice at 200 mg/kg once a day starting at day 2 post-infection and for 21 consecutive days. Control humanized mice received the corresponding vehicle. An appropriate sample size (n=12) was calculated during the study design for each group to obtain a difference on viral load of 15% between the groups by taking into account a common standard deviation of 10% using a bilateral student t-test based on a 95% confidence level. The level of humanization was randomized between the two groups before HIV-1 infection.
6
Inflammasome Activation in MDMi Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For inflammasome activation experiments, MDMi were primed with 200 ng/ml of ultrapure LPS (E.Coli 0111:B4, Invivogen) for 3 h or 50 μg of S-clamp or F-clamp for 6 h. Cells were washed in after priming to remove residual LPS or S-Clamp and cells were stimulated with conventional NLRP3 inflammasome activators ATP (5 mM, Sigma) and nigericin (10 μM, Invivogen), or fibrillar α-synuclein (10 μM, Proteos), S-Clamp (2–50 μg) or SARS-CoV-2 isolates (MOI 0.1, 1) for the indicated time. For priming studies, MDMis were pre-treated with the NF-kB inhibitor, Bay 11-7082 (3 μM, Sigma), before stimulation with S-clamp and the addition of ATP, nigericin or α-synuclein. For inhibition studies, MCC950 (10 μM), VX-765 (20 μM, Invivogen) and MLN-4760 (1,10 μM, Sigma) were added after the priming step. At the end of treatment, the supernatant was collected and stored at −80 °C until analysis by enzyme-linked immunosorbent assay (ELISA) or western blotting.
7
Monocyte Priming and Activation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary monocytes were seeded into 96‐well plates at 5 × 103 cells/well, in RPMI 1640, GlutaMAX medium (Thermo Fisher Scientific) supplemented with 10% fetal calf serum (Lonza). When indicated, primary monocytes were incubated for 3 h in the presence of LPS (10 ng/ml, InvivoGen). Unless otherwise indicated, primary monocytes were then treated for 90 min with nigericin (5 μM, InvivoGen), ATP (2.5 mM, Sigma) (Mariathasan et al, 2006 ), staurosporine (1.25 μM, Tocris), UCN‐01 (12.5 μM, Sigma), or Ro31‐8820 (100 μM, Tocris). When indicated, monocytes were treated with colchicine (1 μM or at the indicated concentration, Sigma), MCC950 (10 μM, Adipogen AG‐CR1‐3615), paclitaxel (Taxol, 5 μM, Sigma), nocodazole (5 μM, Sigma), z‐YVAD‐FMK, z‐IETD‐FMK, z‐DEVD‐FMK (at the indicated concentrations, Bachem, #4027532, #4034771, #4027402, respectively), VX‐765 (InvivoGen), 30 min before addition of UCN‐01, TcdB (Abcam, #ab124001, 125 ng/ml), and TcdA (1 μg/ml) or nigericin. TcdA was purified from Clostridioides difficile VPI10463 strain, as previously described (von Eichel‐Streiber et al, 1987 ; Popoff, 1987 ). Following the incubation, cells were centrifuged and supernatants were collected.
8
Culturing and Differentiating Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary blood cells, THP-1 and U937 cells were cultured in RPMI1640 (Gibco) supplemented with 10% FCS, 2 mM sodium pyruvate and 1 mM l -glutamine. THP-1 cells were derived from a patient with acute monocytic leukaemia and U937 cells are myeloid cells derived from a patient with histiocytic lymphoma. Primary human macrophages were differentiated by magnetically separating CD14-positive cells (BD Biosciences) from peripheral blood mononuclear cells extracted on a Ficoll gradient (GE Healthcare) and subsequently differentiated over 10 days using 50 ng/ml M-CSF (Peprotech). They were stimulated the following day in the presence of 5 ng/ml M-CSF. Differentiation of both THP-1 and U937 cells were carried out using 50 ng/ml PMA for 24 h. Cells were rested for 36–48 h prior to stimulation. HEK293 null 1 (Invivogen) cells are HEK293 cells stably transfected with an NF-κB/AP-1 reporter. They were grown in high-glucose DMEM supplemented with 10% FCS, 2 mM sodium pyruvate and 1 mM l -glutamine, with the addition of 100 μg/ml normocin every third passage. MG-132 was from EMD Millipore, and bortezomib and carfilzomib were from Selleck chemicals. z-YVAD-fmk and z-IETD-fmk were from R&D systems. VX-765 was from Invivogen. AEBSF-HCl was from Enzo Lifesciences.
9
Investigating Cellular Pathways with Diverse Reagents
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following reagents were used: Ivermectin (Sigma I8898-250MG), KN-93 (Tocris, 5215-1 MG), Cyclosporin A (Tocris, 1101–100 MG), KN-62 (Tocris, 1277-1 MG), PSB 069 (Santa Cruz, sc-204216 10 MG), DCPIB (Tocris, 1540–10 MG), A438079 (Tocris, 2972–10 MG), z-vad-fmk (Enzo Life Sciences, ALX-260-020-M001-1 MG), vx765 (InvivoGen, inh-vx765-1-10MG), oxATP (Sigma Aldrich, A6779–25MG), PPAD (Sigma Aldrich, P178–10MG), DPI (Sigma Aldrich, D2926–10 MG), NAC (Sigma Aldrich, A9165–25 MG), Suramin (Sigma Aldrich, S2671–100 MG), Necrostatin (Enzo Life Sciences, BML-AP309–0020–20 MG), Digoxin (Sigma Aldrich, D6003–100 MG), Apyrase (New England Biolabs, M0393S–10,000 milliunits), ATP (Sigma Aldrich, A2383–1G), Pannexin-1-Inhibitor (AnaSpec, 61911–1 MG), Probenecid (Sigma Aldrich, P8761–25 G), YOPRO-1 Iodide (Life Technologies, Y3603-1 mL), GSH (Sigma Aldrich, G4251–1 G).
10
Inhibition of Inflammatory Pathways in THP-1 Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PMA-differentiated, THP-1 macrophages were pre-incubated with the indicated amounts of either the IKK-β inhibitors TPCA-1 (Abcam, Cambridge, UK) or BMS-345541 (Abcam), the unspecific inflammasome inhibitor VX-765 (InvivoGen), which inhibits caspase-1 activity, the specific NLRP3-inflammasome inhibitor MCC950 (InvivoGen), or the MAPK inhibitors SP600125 (SAP/JNK MAPK inhibitor, InvivoGen), SB202190 (p38α/β MAPK inhibitor, InvivoGen), or U0126 (ERK MAPK inhibitor, Cell Signaling Technologies, Leiden, The Netherlands) for 90 min and subsequently stimulated with rFlaA:Betv1 for 24 h. The target molecules of the used inhibitors are summarized in Supplementary Figure 2
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!