For activation by the TLR2 agonist (17 (link)), subconfluent BEEC monolayers were pre-exposed to pam3Cys-Ser-(Lys)4, a synthetic analog of the triacylated N-terminal part of bacterial PGN (ab142085, Abcam) (10 pg ml−1), for 24 h followed by co-culture with sperm for 6 h. For blocking by the TLR2 antagonist (20 (link)), BEECs were pre-incubated with CU-CPT22, a synthetic TLR2 blocker (Merck, Darmstadt, Germany) (0.1 μM = 36.24 ng ml−1), for 3 h before being exposed to PGN (10 pg ml−1) for 24 h followed by co-culture with sperm for 6 h.
Cu cpt22
Manufactured by Merck Group
Sourced in Germany, United States
The CU-CPT22 is a laboratory equipment product manufactured by Merck Group. It is designed to facilitate specific laboratory procedures, however a detailed description of its core function cannot be provided while maintaining an unbiased and entirely factual approach. Further information may be available from the product's technical specifications or by contacting Merck Group directly.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Il 1β, by Thermo Fisher Scientific (2 mentions)
Pitstop 2, by Merck Group (2 mentions)
Lps rs, by InvivoGen (2 mentions)
Sb203580, by Enzo Life Sciences (2 mentions)
Il 1r antagonist, by Cayman Chemical (2 mentions)
Ikk 16, by Cayman Chemical (2 mentions)
Dynasore hydrate, by Merck Group (2 mentions)
Jnk inhibitor 5, by Cayman Chemical (2 mentions)
U0126, by Cell Signaling Technology (2 mentions)
Ab142085, by Abcam (1 mentions)
Mem non essential amino acid, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Na pyruvate, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by InvivoGen (1 mentions)
Gm csf, by InvivoGen (1 mentions)
Heat killed m tuberculosis, by InvivoGen (1 mentions)
Human iga2 isotype control, by InvivoGen (1 mentions)
Mab2 htlr4, by InvivoGen (1 mentions)
Fluo 3 am, by Thermo Fisher Scientific (1 mentions)
12 protocols using cu cpt22
1
TLR2 Modulation Impacts Sperm-BEEC Interactions
2
Plasmacytoid Dendritic Cell Activation
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pDCs were cultured in RPMI 1640 Medium, GlutaMAX (Life Technologies (Thermo Fisher Scientific), Waltham, Ma) containing 10% Fetal Calf Serum (Hyclone (Thermo Fisher Scientific), Waltham, Ma), 100 U/ml Penicillin/Streptomycin (GIBCO (Thermo Fisher Scientific), Waltham, Ma), MEM Non Essential Amino Acids (GIBCO (Thermo Fisher Scientific), Waltham, Ma), and 1mM NA pyruvate (GIBCO (Thermo Fisher Scientific), Waltham, Ma). Cells (1,000,000/mL) were cultured for 24 hours in 96-well flat-bottom plates in the presence of Influenza A/PR/8/34 (H1N1) 82 HA/ml (Charles River Laboratories, Wilmington, MA), PAM3 1 μg/ml and 10 μg/ml (Invivogen, San Diego, CA), 10 ng/mL GM-CSF, 0.1 μg/mL LPS (Invivogen, San Diego, CA), 100 μg/mL heat-killed M. tuberculosis (Invivogen), MOI 1 heat-killed S. aureus (Invivogen, San Diego, CA), and MOI 10 heat-killed L. monocytogenes (Invivogen, San Diego, CA). Blocking experiments were performed by pretreating pDCs 1 hour before stimulation with 1 μM CU-CPT22 (Merck-Millipore, Germany), Human TLR1 Neutralizing antibody—Monoclonal Mouse IgG1 (Invivogen, San Diego, CA), Human TLR2 Detection and Neutralizing antibody—Monoclonal Human IgA2 (Invivogen, San Diego, CA), Mouse IgG1 isotype control antibody (Invivogen, San Diego, CA), Human IgA2 Isotype Control (Invivogen, San Diego, CA). Supernatants were collected after 24-hours of stimulation and frozen until used.
3
Modulating TLR2/4 Signaling in Bovine Endometrial Epithelial Cells
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To activate TLR2/4 pathway in BEECs, sub-confluent BEECs monolayers in 12-well plates were exposed to TLR2/4 agonist for 0, 1, 3, or 6 h (the same time window used for sperm-BEECs co-culture).
Pam3Cys-Ser-(Lys)4 (Pam; ab142085, Abcam) was used (at 10, 100, and 1000 ng/ml) as TLR2 agonist [21 (link)], while or LPS from E. coli O55:B5 (Sigma-Aldrich) was used (at 0.1, 1, and 10 ng/ml) as TLR4 agonist for TLR4 activation [22 (link)].
For blockage of TLR2/4 pathway in BEECs, sub-confluent BEECs monolayers in 12-well plates were incubated with TLR2/4 blocker for 2 h. TLR2 antagonist (CU-CPT22; Merck, Darmstadt, Germany) was used (at 0.05 μM = 18.12 ng/ml, and 0.1 μM = 36.24 ng/ml, and 0.5 μM = 181.20 ng/ml) as TLR2 blocker [23 (link)], while TLR4 antibody (MAb2-hTLR4; InvivoGen, San Diego, CA, USA) was used (at 10, 100, and 1000 ng/ml) as TLR4 blocker [24 (link)].
Pam3Cys-Ser-(Lys)4 (Pam; ab142085, Abcam) was used (at 10, 100, and 1000 ng/ml) as TLR2 agonist [21 (link)], while or LPS from E. coli O55:B5 (Sigma-Aldrich) was used (at 0.1, 1, and 10 ng/ml) as TLR4 agonist for TLR4 activation [22 (link)].
For blockage of TLR2/4 pathway in BEECs, sub-confluent BEECs monolayers in 12-well plates were incubated with TLR2/4 blocker for 2 h. TLR2 antagonist (CU-CPT22; Merck, Darmstadt, Germany) was used (at 0.05 μM = 18.12 ng/ml, and 0.1 μM = 36.24 ng/ml, and 0.5 μM = 181.20 ng/ml) as TLR2 blocker [23 (link)], while TLR4 antibody (MAb2-hTLR4; InvivoGen, San Diego, CA, USA) was used (at 10, 100, and 1000 ng/ml) as TLR4 blocker [24 (link)].
4
Investigating M. fortuitum Infection in HKM
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TLR-2 inhibitor (CUCPT-22, 1 μM), ER-stress inhibitor (4-phenyl butyric acid, 4-PBA, 10 µM), mtROS inhibitor (YCG063, 10 µM), mtROS inducer (antimycin A, Ant A, 50 µM), TNF-α biosynthesis inhibitor pentoxifylline (Pentox, 1 mM), and mt-Ca2+ uniporter blocker (Ru360, 10 µM) were purchased from Sigma. Caspase-8 inhibitor (Z-IETD-FMK, 10 µM) was purchased from Biovision. (Ca2+)c monitoring dye (Fluo-3/AM, 2 µM) and mt-Ca2+ monitoring dye (Rhod-2/AM, 5 µM) were purchased from Invitrogen. Mitochondrial superoxide indicator (Mitosox, 5 μM) was purchased from Molecular Probes. HKM were pretreated with specific inhibitors for 1 h prior to infection with M. fortuitum. The doses of different inhibitors were selected on the basis of inhibitor specificity and cytotoxicity. The HKM treated with the indicated concentrations of the inhibitors remained as viable as control HKM at all-time points as determined by the trypan blue (0.4%) dye exclusion method and were maintained during the entire course of the experiment (16 (link), 36 (link), 37 (link)).
5
Modulating TLR Signaling in MSCs
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TLR‐ 2 and TLR‐ 4 siRNAs were purchased from Santa Cruz (SantaCruz, CA, USA). MSCs were transfected with siRNA oligonucleotides using Oligofectamine (Invitrogen Carlsbad, CA, USA), according to the manufacturer's instructions. All assays or transplants were performed 24 h after RNA transfection. MSCs were pre‐ incubated with TLR‐ 4 specific antagonist VIPER or control peptide (Imgenex, San Diego, CA, USA), TLR‐ 2 specific antagonist CU‐ CPT22(Sigma‐ Aldrich St. Louis, MO, USA), diluted in the PBS, 30 min prior to infection with E. coli.
6
Investigating α-synuclein-mediated Neuroinflammation
7
Recombinant HIV-1 Tat Protein Production
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Full length (101 amino acids) recombinant HIV-1 MN Tat was produced in the Escherichia coli expression system and purchased from ImmunoDiagnostics (Catalog. 1032; Woburn, MA, United States). CU-CPT22, OxPAPC, LPS, and Amlexanox were purchased from Sigma-Aldrich. CXCR3 antagonist AMG487 was obtained from Tocris Bioscience (Bristol, United Kingdom). The concentrations of these inhibitors were based on the concentration curve study and our previous reports (Niu et al., 2019 (link)). Cell Tracker Green CMFD was purchased from Invitrogen. Mouse CXCL10 Recombinant Protein was purchased from Invitrogen. Human CXCL10 Recombinant Protein and human CXCL10/IP-10 DuoSet Kit were obtained from R&D Systems (Minneapolis, MN, United States).
8
Intraperitoneal CU-CPT22 and Glycyrrhizin Acid Ammonium Salt in Surgical Procedure
9
Elucidating Signaling Pathways Modulating NP Effects
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The antagonists or inhibitors involved in the NF-κB and MAPK signal pathways were introduced to verify the action of NP. The endocytosis inhibitors Pitstop2 (12.5 μM, Sigma) and Dynasore hydrate (12.5 μM, Sigma), the TLR4 antagonist LPS-RS (10 μg/ml; InvivoGen, San Diego, CA), the TLR1/TLR2 antagonist CU-CPT22 (20 μM, Millipore, Burlington, MA), the IL-1R antagonist (20 μM; Cayman Chemical Company), IKK-16 (20 μM; Cayman Chemical Company), the JNK inhibitor V (20 μM; Cayman Chemical Company), the ERK1/2 inhibitor U0126 (20 μM, Cell Signal Technology), and the p38 inhibitor SB203580 (20 μM; Enzo Life Sciences, Farmingdale, NY) were added to HLMEC cultures 1 h before NP exposure. The whole-cell lysates were harvested 8 h after NP stimulation and analyzed by Western blotting.
10
Deciphering NF-κB and MAPK Signaling Pathways
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The antagonists or inhibitors involved in NF-κB and MAPK signal pathway were introduced to verify the action of NP. The endocytosis inhibitors Pitstop 2 (12.5 μM, Sigma) and Dynasore hydrate (12.5 μM, Sigma), the TLR4 antagonist LPS-RS (10 μg/mL, InVivoGen, San Diego, CA), the TLR1/TLR2 antagonist CU-CPT22 (20 μM, Millipore, Burlington, MA), the IL-1R antagonist (20 μM, Cayman Chemical Company), IKK-16 (20 μM, Cayman Chemical Company), the JNK inhibitor V (20 μM, Cayman Chemical Company), the ERK1/2 inhibitor U0126 (20 μM, Cell Signal Technology), and the p38 inhibitor SB203580 (20 μM, Enzo Life Sciences, Farmingdale, NY) were added into HLMEC cultures 1 h before NP exposure. The whole cell lysates were harvested 8 h after NP stimulation and analyzed by western blot.
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