Immunopeptidomics:Article Title: The inflammatory architecture reflects the effects of pharmacological and genetic interventions on resolution of TLR2-mediated inflammation
Article Snippet: .. CD8a FITC 1:100 BD Pharmingen 53-6.7 553030 CD11b FITC 1:400 BioRad M1/70.15 MCA74F CD11c PE 1:100 Miltenyi Biotec N418 130-122-952 CD19 FITC 1:100 BDPharmingen 1D3 557398 CD22 APC 1:100 Miltenyi Biotec Cy34.1 130-102-576 CD27 PE 1:100 Miltenyi Biotec REA499 130-114-166 CD31 PE 1:400 BD Biosciences MEC13.3 553373 CD45 FITC 1:200 Miltenyi Biotec 30F11.1 130-116-535 CD41 FITC 1:50 AbDSerotec MWReg30 MCA2245F CD80 FITC 1:100 Biolegend 16-10A1 104706 CD86 PE 1:100 Biolegend GL-1 105008 CD117 APC 1:50 Bioss bs-10005R-Cy5 CD206 APC 1:100 Biolegend C068C2 141708 CD183 (CXCR3) PE 1:100 Miltenyi Biotec REA724 130-111-087 CD194 (CCR4) PE 1:100 Biolegend 2G12 131203 CD196 (CCR6) PE 1:100 Biolegend 29-2L17 129803 Cytokeratin AF488 1:800 eBiosciences AE1/AE3 53-9003-82 CX3CR1 FITC 1:200 Biolegend SA011F11 149020 CXCR7 APC 1:50 Biolegend 8F11-M16 331113 F4-80 PE 1:400 Biolegend BM8 123110 Ly6C APC 1:100 eBioscience HK 1.4 17-5932-82 Ly6G-Gr1 PE 1:400 eBioscience RB6-8C5 12-5931-83 Ly6G APC 1:100 Biolegend 1A8 127614 MHC II PE 1:100 Miltenyi Biotec REA813 130-102-186 Propidium-Iodide 1:800 Sigma P4170 Siglec F PE 1:200 BD Bioscience E50-2440 552126 ..
Article Title: The inflammatory architecture reflects the effects of pharmacological and genetic interventions on resolution of TLR2-mediated inflammation
Article Snippet: .. Target Fluoro- chrome Dilution Company Clone number Order number CCR10 PE 1:50 R+D Systems FAB2815P CD3 APC 1:100 Miltenyi Biotec REAG1 130-109-838 CD4 AF488 1:100 Southern Biotech L3T4 1540-02 CD8a FITC 1:100 BD Pharmingen 53-6.7 553030 CD11b FITC 1:400 BioRad M1/70.15 MCA74F CD11c PE 1:100 Miltenyi Biotec N418 130-122-952 CD19 FITC 1:100 BDPharmingen 1D3 557398 CD22 APC 1:100 Miltenyi Biotec Cy34.1 130-102-576 CD27 PE 1:100 Miltenyi Biotec REA499 130-114-166 CD31 PE 1:400 BD Biosciences MEC13.3 553373 CD45 FITC 1:200 Miltenyi Biotec 30F11.1 130-116-535 CD41 FITC 1:50 AbDSerotec MWReg30 MCA2245F CD80 FITC 1:100 Biolegend 16-10A1 104706 CD86 PE 1:100 Biolegend GL-1 105008 CD117 APC 1:50 Bioss bs-10005R-Cy5 CD206 APC 1:100 Biolegend C068C2 141708 CD183 (CXCR3) PE 1:100 Miltenyi Biotec REA724 130-111-087 CD194 (CCR4) PE 1:100 Biolegend 2G12 131203 CD196 (CCR6) PE 1:100 Biolegend 29-2L17 129803 Cytokeratin AF488 1:800 eBiosciences AE1/AE3 53-9003-82 CX3CR1 FITC 1:200 Biolegend SA011F11 149020 CXCR7 APC 1:50 Biolegend 8F11-M16 331113 F4-80 PE 1:400 Biolegend BM8 123110 Ly6C APC 1:100 eBioscience HK 1.4 17-5932-82 Ly6G-Gr1 PE 1:400 eBioscience RB6-8C5 12-5931-83 Ly6G APC 1:100 Biolegend 1A8 127614 MHC II PE 1:100 Miltenyi Biotec REA813 130-102-186 Propidium-Iodide 1:800 Sigma P4170 Siglec F PE 1:200 BD Bioscience E50-2440 552126 Antibodies used only for FACS Analysis CD11c BV711 1:50 Miltenyi 130-102-797 CD11b BV605 1:50 Biolegend M1/70 101237 CD45 AF700 1:50 Biolegend 30-F11 103128 CD86 PE 1:50 Biolegend GL-1 105032 F4-80 FITC 1:50 Miltenyi REA126 130-117-509 Ly6G APC-Cy7 1:50 Biolegend 1A8 127624 Siglec F PE- Vio770 1:50 Miltenyi ES22-10D8 130-102-167 Figure S2: Binding of the anti-Ly6G antibody 1A8 does not interfere with GR-1 antibody binding. ..
Incubation:Article Title: Deletion of TP signaling in macrophages delays liver repair following APAP-induced liver injury by reducing accumulation of reparative macrophage and production of HGF.
Article Snippet: .. The sections were incubated with a rabbit anti-mouse TP (1:100; Cayman Chemical, Ann Arbor, MI, USA), a rabbit anti-mouse TXS(1:100; Bioss, Woburn, MA, USA), a rat anti-mouse CD68 antibody (1:100; Bio-Rad Laboratories, Hercules, CA, USA), a rat anti-mouse CD41 (BioRad Laboratories), and a rabbit anti-mouse HGF (1:100; Proteintech Group, Rosemont, Il, US,) at 4°C overnight. .. The sections were then incubated with the following secondary antibodies at 4°C for 1 h: Alexa Fluor 488-conjugated donkey anti-rabbit IgG, Alexa Fluor 594-conjugated donkey anti-rabbit IgG, or Alexa Fluor 647-conjugated donkey anti-rabbit IgG (Molecular Probes, Eugene, OR, USA).
Article Title: Deletion of TP signaling in macrophages delays liver repair following APAP-induced liver injury by reducing accumulation of reparative macrophage and production of HGF
Article Snippet: .. The sections were incubated with a rabbit anti-mouse TP (1:100; Cayman Chemical, Ann Arbor, MI, USA), a rabbit anti-mouse TXS(1:100; Bioss, Woburn, MA, USA), a rat anti-mouse CD68 antibody (1:100; Bio-Rad Laboratories, Hercules, CA, USA), a rat anti-mouse CD41 (BioRad Laboratories), and a rabbit anti-mouse HGF (1:100; Proteintech Group, Rosemont, Il, US,) at 4°C overnight. .. The sections were then incubated with the following secondary antibodies at 4°C for 1 h: Alexa Fluor 488-conjugated donkey anti-rabbit IgG, Alexa Fluor 594-conjugated donkey anti-rabbit IgG, or Alexa Fluor 647-conjugated donkey anti-rabbit IgG (Molecular Probes, Eugene, OR, USA).
Article Title: Physiological platelet aggregation assay to mitigate drug-induced thrombocytopenia using a microphysiological system
Article Snippet: .. Then, 4% PFA (100 μL/well) was added into 96-well cell culture plates and incubated at room temperature for 20 min. After washing thrice with PBS, 1.5% BSA in PBS (100 μL) was added and allowed to stand at room temperature for 30 min. Then, an anti-CD41 antibody with 1.5% BSA in PBS (100 μL of 10 μg/mL; MCA2245; Bio-Rad Laboratories, Hercules, CA, USA) was added and allowed to stand at room temperature for 60 min. After washing thrice with PBS, an anti-rat secondary antibody conjugated to Alexa FluorTM488 with 1.5% BSA in PBS (100 μL of 5 μg/mL; A-11006; Thermo Fisher Scientific, Waltham, MA, USA) was added and allowed to stand at room temperature for 60 min. After washing thrice with PBS, fluorescent images were captured using an IN Cell Analyzer 6500 HS (General Electric Life Sciences/Cytiva). ..
Chromatin Immunoprecipitation:Article Title: Critical role for platelet Ral GTPases in regulating venous thrombosis in mice.
Article Snippet: .. Fluorescein isothiocyanate-conjugated CD41 antibody was from BioRad (#MCA2245F); phycoerythrin-conjugated CD45 antibody was from BioLegend (#30-F11); anti-Ly-6G was from Thermo Fisher Scientific (#RB6-8C5); anti–P-selectin antibody was from Santa Cruz Biotechnology (#sc-6942); anti-histone H3 (citrulline R2 + R8 + R17, ChIP grade, #ab5103) was from Abcam. .. Alexa Fluor-conjugated secondary antibodies were from Thermo Fisher Scientific.
Flow Cytometry:Article Title: Platelet-specific SLFN14 deletion causes macrothrombocytopenia and platelet dysfunction through dysregulated megakaryocyte and platelet gene expression
Article Snippet: The prepared libraries were quantified via a fluorometric method involving a Promega QuantiFluor dsDNA assay and qualified using electrophoretic separation on the Agilent TapeStation 4200. .. Supplemental Table 1: Antibodies used in flow cytometry Antibody Host species, conjugate, catalogue number Manufacturer Dilution CD41 (αIIb) – mouse Rat, APC, 133913 Biolegend FCC 1:200 FC 1:100 CLEC2 - mouse Rat, FITC, 17D9MCA5700F Bio-Rad Laboratories FC 1:100 CD42a (GpIb) – mouse Rat, FITC, M040-1 Emfret Analytics FC 1:100 CD49b (α2) – mouse Rat, FITC, M071-1 Emfret Analytics FC 1:100 CD41/CD61 (integrin αIIbβ3) – mouse Rat, FITC, M025-1 Emfret Analytics FC 1:100 GPVI – mouse Rat, FITC, M011-1 Emfret Analytics FC 1:100 CD62P (P-selectin) – mouse Rat, FITC, M130-1 Emfret Analytics FC 1:100 CD41/CD61 activated (JON/A) – mouse Rat, PE, M023-2 Emfret Analytics FC 1:100 Isotype controls IgG Rat, FITC, P190-1 Emfret Analytics FC 1:100 IgG2a Rat, PE, 400507 Biolegend FC 1:100 IgG1 Rat, APC, 400411 Biolegend FC 1:100 IgG2b Rat, FITC, MCA6006F Bio-Rad Laboratories FC 1:100 IgG2b Rat, APC e-780, 15321650 eBioscience FC 1:100 Supplemental Table 2: Platelet agonists Agonist Receptor interaction Manufacturer, catalogue number ADP P2Y1, P2Y12 Sigma, A2754 Collagen GPVI, αIIbβ3 Takeda, 1130630 CRP (collagen related peptide) GPVI Provided by Prof. Farndale; cross-linked in-house PAR4-peptide PAR4 Alta biosciences Thrombin PAR1, PAR4 (PAR3 and PAR4 in mice) Sigma, T4648 U46619 TP (thromboxane A2) Sigma, D8174 Supplementary figure 1: Chi square analyses to assess for Mendelian inheritance patterns shows both (i) flox and (ii) PF4Cre alleles were inherited within Mendelian ratios. ..
FACS:Article Title: The inflammatory architecture reflects the effects of pharmacological and genetic interventions on resolution of TLR2-mediated inflammation
Article Snippet: .. Target Fluoro- chrome Dilution Company Clone number Order number CCR10 PE 1:50 R+D Systems FAB2815P CD3 APC 1:100 Miltenyi Biotec REAG1 130-109-838 CD4 AF488 1:100 Southern Biotech L3T4 1540-02 CD8a FITC 1:100 BD Pharmingen 53-6.7 553030 CD11b FITC 1:400 BioRad M1/70.15 MCA74F CD11c PE 1:100 Miltenyi Biotec N418 130-122-952 CD19 FITC 1:100 BDPharmingen 1D3 557398 CD22 APC 1:100 Miltenyi Biotec Cy34.1 130-102-576 CD27 PE 1:100 Miltenyi Biotec REA499 130-114-166 CD31 PE 1:400 BD Biosciences MEC13.3 553373 CD45 FITC 1:200 Miltenyi Biotec 30F11.1 130-116-535 CD41 FITC 1:50 AbDSerotec MWReg30 MCA2245F CD80 FITC 1:100 Biolegend 16-10A1 104706 CD86 PE 1:100 Biolegend GL-1 105008 CD117 APC 1:50 Bioss bs-10005R-Cy5 CD206 APC 1:100 Biolegend C068C2 141708 CD183 (CXCR3) PE 1:100 Miltenyi Biotec REA724 130-111-087 CD194 (CCR4) PE 1:100 Biolegend 2G12 131203 CD196 (CCR6) PE 1:100 Biolegend 29-2L17 129803 Cytokeratin AF488 1:800 eBiosciences AE1/AE3 53-9003-82 CX3CR1 FITC 1:200 Biolegend SA011F11 149020 CXCR7 APC 1:50 Biolegend 8F11-M16 331113 F4-80 PE 1:400 Biolegend BM8 123110 Ly6C APC 1:100 eBioscience HK 1.4 17-5932-82 Ly6G-Gr1 PE 1:400 eBioscience RB6-8C5 12-5931-83 Ly6G APC 1:100 Biolegend 1A8 127614 MHC II PE 1:100 Miltenyi Biotec REA813 130-102-186 Propidium-Iodide 1:800 Sigma P4170 Siglec F PE 1:200 BD Bioscience E50-2440 552126 Antibodies used only for FACS Analysis CD11c BV711 1:50 Miltenyi 130-102-797 CD11b BV605 1:50 Biolegend M1/70 101237 CD45 AF700 1:50 Biolegend 30-F11 103128 CD86 PE 1:50 Biolegend GL-1 105032 F4-80 FITC 1:50 Miltenyi REA126 130-117-509 Ly6G APC-Cy7 1:50 Biolegend 1A8 127624 Siglec F PE- Vio770 1:50 Miltenyi ES22-10D8 130-102-167 Figure S2: Binding of the anti-Ly6G antibody 1A8 does not interfere with GR-1 antibody binding. ..
Binding Assay:Article Title: The inflammatory architecture reflects the effects of pharmacological and genetic interventions on resolution of TLR2-mediated inflammation
Article Snippet: .. Target Fluoro- chrome Dilution Company Clone number Order number CCR10 PE 1:50 R+D Systems FAB2815P CD3 APC 1:100 Miltenyi Biotec REAG1 130-109-838 CD4 AF488 1:100 Southern Biotech L3T4 1540-02 CD8a FITC 1:100 BD Pharmingen 53-6.7 553030 CD11b FITC 1:400 BioRad M1/70.15 MCA74F CD11c PE 1:100 Miltenyi Biotec N418 130-122-952 CD19 FITC 1:100 BDPharmingen 1D3 557398 CD22 APC 1:100 Miltenyi Biotec Cy34.1 130-102-576 CD27 PE 1:100 Miltenyi Biotec REA499 130-114-166 CD31 PE 1:400 BD Biosciences MEC13.3 553373 CD45 FITC 1:200 Miltenyi Biotec 30F11.1 130-116-535 CD41 FITC 1:50 AbDSerotec MWReg30 MCA2245F CD80 FITC 1:100 Biolegend 16-10A1 104706 CD86 PE 1:100 Biolegend GL-1 105008 CD117 APC 1:50 Bioss bs-10005R-Cy5 CD206 APC 1:100 Biolegend C068C2 141708 CD183 (CXCR3) PE 1:100 Miltenyi Biotec REA724 130-111-087 CD194 (CCR4) PE 1:100 Biolegend 2G12 131203 CD196 (CCR6) PE 1:100 Biolegend 29-2L17 129803 Cytokeratin AF488 1:800 eBiosciences AE1/AE3 53-9003-82 CX3CR1 FITC 1:200 Biolegend SA011F11 149020 CXCR7 APC 1:50 Biolegend 8F11-M16 331113 F4-80 PE 1:400 Biolegend BM8 123110 Ly6C APC 1:100 eBioscience HK 1.4 17-5932-82 Ly6G-Gr1 PE 1:400 eBioscience RB6-8C5 12-5931-83 Ly6G APC 1:100 Biolegend 1A8 127614 MHC II PE 1:100 Miltenyi Biotec REA813 130-102-186 Propidium-Iodide 1:800 Sigma P4170 Siglec F PE 1:200 BD Bioscience E50-2440 552126 Antibodies used only for FACS Analysis CD11c BV711 1:50 Miltenyi 130-102-797 CD11b BV605 1:50 Biolegend M1/70 101237 CD45 AF700 1:50 Biolegend 30-F11 103128 CD86 PE 1:50 Biolegend GL-1 105032 F4-80 FITC 1:50 Miltenyi REA126 130-117-509 Ly6G APC-Cy7 1:50 Biolegend 1A8 127624 Siglec F PE- Vio770 1:50 Miltenyi ES22-10D8 130-102-167 Figure S2: Binding of the anti-Ly6G antibody 1A8 does not interfere with GR-1 antibody binding. ..
Cell Culture:Article Title: Physiological platelet aggregation assay to mitigate drug-induced thrombocytopenia using a microphysiological system
Article Snippet: .. Then, 4% PFA (100 μL/well) was added into 96-well cell culture plates and incubated at room temperature for 20 min. After washing thrice with PBS, 1.5% BSA in PBS (100 μL) was added and allowed to stand at room temperature for 30 min. Then, an anti-CD41 antibody with 1.5% BSA in PBS (100 μL of 10 μg/mL; MCA2245; Bio-Rad Laboratories, Hercules, CA, USA) was added and allowed to stand at room temperature for 60 min. After washing thrice with PBS, an anti-rat secondary antibody conjugated to Alexa FluorTM488 with 1.5% BSA in PBS (100 μL of 5 μg/mL; A-11006; Thermo Fisher Scientific, Waltham, MA, USA) was added and allowed to stand at room temperature for 60 min. After washing thrice with PBS, fluorescent images were captured using an IN Cell Analyzer 6500 HS (General Electric Life Sciences/Cytiva). ..
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![18 Fr puncture of hemostasis in porcine aorta using VWP by validating the memory programming effect of each part. a, As a challenging model for application of large-diameter catheters, i) an 18 Fr (6 mm) puncture is created into the porcine thoracic aorta (diameter: 10 mm) so that the size-matched VWP is deployed, followed by measuring proximal and distal blood pressure. ii) The experimental groups are designed first to exam the memory programming effect of collaboration between Ring squeezing with Body expansion on self-locking (SL) to enable efficient hemostasis. Next, the effect of Wing shape recovery from curve to flat is examined on hemodynamic control (HC) in cooperation with the actions of Body and Ring to handle hemostasis. No recovery of Wing shape is expected to induce excessive thrombosis. iii) Four experimental groups are established using a total of 12 pigs (N = 12) with immediate sacrifice following deployment (N = 3 each). Group 1 [SL(−) HC(−)] represents no memory programming. Group 2 [SL(+) w/flat Wing] has the effects of Body and Ring actions except the hemostatic sealing by keeping the flat Wing. Group 3 [SL(+) HC(+)] possesses the complete memory effects of the three parts. Group 4 [SL(+) w/bump Wing] is expected to have excessive thrombosis because of no shape recovery from the curved Wing while maintaining the memory actions of Body and Wing. b, Each group is visually explained in the illustrations. c , In VWP actions, (left) the bleeding condition preserves the normal sinusoidal waveform of high proximal pressure (green) in contrast to the disturbed waveform of low distal pressure (red). (middle) Hemostatic closure results in similar high sinusoidal waveform at both pressure sites. (right) Excessive thrombosis does not disturb the waveform, but the distal pressure level becomes lower than the proximal one. d, When reperfusion starts by removing the clamp post-deployment (blue), only Group 3 [SL(+) HC(+)] reaches the hemostatic closure, as evidenced by flow stabilization (red) with a 5 s plateau at both pressure sites. Group 4 [SL(+) w/bump Wing] exhibits the pattern of over-thrombosis. e, H&E images show bleeding in Group 1 as an indication of incomplete closure in contrast to moderate, minimal, and dense thrombotic features observed in Group 2, 3, and 4 respectively as further supported by the signals of activated platelets (green, <t>CD41-positive)</t> and fibrinogen (red) [Scale bars = 0.5 mm (4 mm in box)]. f, Compared to Group 1 [SL(−) HC(−)] and 2 [SL(+) w/flat Wing], Group 3 [SL(+) HC(+)] shows the fastest i) hemostasis and ii) arterial pressure equilibration, indicating the most efficient hemostatic response. g, These outcomes in Group 3 include i) the smallest difference between the proximal and distal pressures with ii) the smallest thrombus area in contrast the largest area of Group 4 [SL(+) w/bump Wing] as an indication of excessive thrombosis. h , The marker gene expression of thrombotic feature (vWF, PF-4, and P-sel) significantly increases from Group 2 to Group 3 and further to Group 4 except the comparison of vWF expression between Group 2 and 3 (ns: no significance). Data are shown as mean ± SD, N = 3 biologically independent animals per group. Significance was determined using one-way ANOVA with Tukey's test between groups.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_3994/pmc12803994/pmc12803994__gr4.jpg)