fitc conjugated anti cd41  (4A Biotech)

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: Phosphatidylserine-blocking nanoparticles inhibit thrombosis without increased bleeding in mice

doi: 10.1016/j.jtha.2024.10.007

Figure Lengend Snippet: ( A ) Whole blood cells from freshly drawn citrated human blood were precipitated and washed to deplete fibrinogen, and DPAL was added to cell suspensions at 6 x 10 11 particles/mL, followed by stimulation with thrombin (Thr) and/or convulxin (CVX) in the presence of 2 mM CaCl 2 as indicated. Platelets were identified in a flow cytometer by forward and side scatter, and counterstaining with AlexaFluor ® 488- or AlexaFluor ® 647-conjugated anti-CD41 antibodies. (i) Liposome binding was assessed by Cy3 fluorescencein CD41+ platelets, and are shown as percent Cy3-positive (DPAL+) platelets ± S.E.M. (ii) PS exposure was assessed with Annexin V-FITC (AXV) in separate tubes containing aliquots from the treated samples from (i), shown as mean fluorescence intensities (MFI) ± S.E.M. p < 0.01 for all samples compared to unstimulated. ( B ) (i) Platelets as in (A) were stimulated with thrombin with or without 10 μL AXV added as indicated, and DPAL binding is shown as fold change from unstimulated ± S.E.M. (ii) AXV MFI from the samples in (i) are shown ± S.E.M. ( C ) Washed platelets from WT or Ano6 KO mice were suspended in Tyrode’s buffer in the presence of DPAL at 6 x 10 11 particles/mL, stimulated with 0.1 U/mL thrombin as indicated, and assessed for (i) liposome binding as in (B), using FITC-conjugated anti-Cd41 antibodies to identify murine platelets, or (ii) AXV MFI, shown ± S.E.M. ( D ) Human platelets treated as in (A) were counterstained with FITC-conjugated PAC-1; FITC-PAC-1 MFI are shown ± S.E.M. n.s., no significant differences between groups. (E) Aggregation of human platelets suspended in buffer at 2x10 8 platelets/mL, following stimulation with thrombin at the indicated concentrations was measured in the absence (control, CTL) or presence of DPAL added as in (D). Representative of 4 independent experiments. ( F ) Dilute prothrombin time in pooled normal plasma with added rabbit brain tissue thromboplastin, in the presence or absence of DPAL as indicated, and shown as mean ± S.E.M.

Article Snippet: Rat-anti-mouse Cd41 FITC-conjugated antibodies and dry chemicals were from Fisher Scientific (Pittsburgh, PA, USA).

Techniques: Binding Assay, Flow Cytometry, Fluorescence, Control, Clinical Proteomics

Journal: Journal of Pharmaceutical Analysis

Article Title: Oxymatrine, a novel TLR2 agonist, promotes megakaryopoiesis and thrombopoiesis through the STING/NF-κB pathway

doi: 10.1016/j.jpha.2024.101054

Figure Lengend Snippet: Oxymatrine promotes typical megakaryocyte (MK) differentiation in concentration-dependent manner. (A) Analysis of CD41 + /CD42b + complex surface expression of oxymatrine (10, 20, and 40 μM) and phorbol 12-myristate 13-acetate (PMA; 0.8 nM) intervened K-562 and Meg-01 cell for five days by flow cytometry. (B) The percentage of CD41 + /CD42b + cells in the control and oxymatrine-treated groups ( n = 3 per group). (C) The DNA ploidy analysis of oxymatrine (10, 20, and 40 μM) and PMA (0.8 nM) induced K-562 and Meg-01 cell for five days by flow cytometry. (D) The histogram represents the proportion of total 2 N, 4 N and ≥4 N ploidy in both K-562 and Meg-01 cells ( n = 3 per group). (E) Giemsa staining of K562 and Meg-01 cells with or without oxymatrine (10, 20, and 40 μM) treatment for five days (PMA (0.8 nM) as positive control). (F, G) Phalloidin-labeled cytospin in K562 (F) and Meg-01 (G) cells intervened with multiple concentrations of oxymatrine (10, 20, and 40 μM) for five days under a fluorescence microscope ( n = 3 per group). Excitation wavelength: 560 nm for phalloidin and 405 nm for 4′,6-diamidino-2-phenylindole (DAPI). Data represent the mean ± standard deviation (SD) of three independent experiments and are analyzed by one-way analysis of variance (ANOVA) with Dunnett's 154. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 vs. the control. FITC: fluorescein isothiocyanate; PE: phycoerythrin; PI: propidium iodide; MFI: mean fluorescence intensity.

Article Snippet: The cells were then treated with 8 μM fluorescein isothiocyanate (FITC)-conjugated anti-CD41 (Beijing 4A Biotech Co., Ltd., Beijing, China) and 8 μM phycoerythrin (PE)-conjugated anti-CD42b (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) in 100 μL of PBS.

Techniques: Concentration Assay, Expressing, Flow Cytometry, Control, Staining, Positive Control, Labeling, Fluorescence, Microscopy, Standard Deviation

Journal: Journal of Pharmaceutical Analysis

Article Title: Oxymatrine, a novel TLR2 agonist, promotes megakaryopoiesis and thrombopoiesis through the STING/NF-κB pathway

doi: 10.1016/j.jpha.2024.101054

Figure Lengend Snippet: Oxymatrine administration promotes thrombopoiesis in Tg (cd41:enhanced green fluorescent protein (eGFP)) transgenic zebrafish. (A) Cd41:eGFP + thrombocytes in the caudal hematopoietic tissue (CHT) region of oxymatrine (10, 20, and 40 μM) treated and control zebrafish. (B) Quantification of cd41:eGFP + cells in the CHT region in each group ( n = 4 per group). (C) Flow cytometry analysis of cd41:eGFP + cells in zebrafish embryos. (D) Quantification of cd41:eGFP + cells in the zebrafish embryos in each group ( n = 3 per group). Data represent the mean ± standard deviation (SD) of four or three independent experiments and are analyzed by one-way analysis of variance (ANOVA) with Dunnett's 154. ∗∗ P < 0.01, and ∗∗∗ P < 0.001 vs. the control. SSC: side scatter; FITC: fluorescein isothiocyanate.

Article Snippet: The cells were then treated with 8 μM fluorescein isothiocyanate (FITC)-conjugated anti-CD41 (Beijing 4A Biotech Co., Ltd., Beijing, China) and 8 μM phycoerythrin (PE)-conjugated anti-CD42b (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) in 100 μL of PBS.

Techniques: Transgenic Assay, Control, Flow Cytometry, Standard Deviation

Journal: Journal of Pharmaceutical Analysis

Article Title: Oxymatrine, a novel TLR2 agonist, promotes megakaryopoiesis and thrombopoiesis through the STING/NF-κB pathway

doi: 10.1016/j.jpha.2024.101054

Figure Lengend Snippet: Oxymatrine accelerates platelet recovery in acute X-ray irradiated mice. (A) The strategies of establishing the irradiated mice and treatment of each groups. (B) Peripheral platelet level of control group, thrombopoietin (TPO)-treated group (3000 U/kg), oxymatrine-treated group (20 mg/kg), and model group. Histology analysis was performed on days 0, 4, 7, 10, and 12 ( n = 6 per group). (C) Mean platelet volume (MPV) values in each groups ( n = 6 per group). (D) Leukocyte counts in each group ( n = 6 per group). (E) Erythrocyte counts in each group ( n = 6 per group). (F) Flow cytometry analysis of expression of CD41 and CD61 in peripheral blood (PB) platelets in each group after treatment for 12 days. (G) The histogram exhibits the percentage of expression of CD41 + /CD61 + in PB cells in each group ( n = 3 per group). Data represent the mean ± standard deviation (SD). Two-way analysis of variance (ANOVA) with Tukey's multiple comparisons test was used unless otherwise specified. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 vs. the corresponding model group. IR: ionizing radiation; FITC: fluorescein isothiocyanate; UL: upper left; UR: upper right; LL: lower left; LR: lower right.

Article Snippet: The cells were then treated with 8 μM fluorescein isothiocyanate (FITC)-conjugated anti-CD41 (Beijing 4A Biotech Co., Ltd., Beijing, China) and 8 μM phycoerythrin (PE)-conjugated anti-CD42b (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) in 100 μL of PBS.

Techniques: Irradiation, Control, Flow Cytometry, Expressing, Standard Deviation

Journal: Journal of Pharmaceutical Analysis

Article Title: Oxymatrine, a novel TLR2 agonist, promotes megakaryopoiesis and thrombopoiesis through the STING/NF-κB pathway

doi: 10.1016/j.jpha.2024.101054

Figure Lengend Snippet: Oxymatrine rescues megakaryopoiesis in the bone marrow (BM) and spleen after acute radiation injury. (A) Representative images of hematoxylin and eosin (H&E) staining of BM from control group, model group, thrombopoietin (TPO)-treated (3000 U/kg) group, and oxymatrine-treated (20 mg/kg) group after treatment for 12 days. (B) The histogram exhibits the counts of BM megakaryocyte (MK) in each group ( n = 3 per group). (C) Representative images of H&E staining of spleens from each group after treatment for 12 days. (D) The histogram shows the counts of spleen MKs in each group ( n = 3 per group). (E) The analysis of c-Kit + /CD41 − (hematopoietic progenitor cells), c-Kit + /CD41 + (megakaryocytic progenitor cells), and c-Kit - /CD41 + (MKs) cell expression in each group after treatment for 12 days performed by flow cytometry. (F) The histogram exhibits the percentage of c-Kit + /CD41 − , c-Kit + /CD41 + , and c-Kit - /CD41 + cells in each group ( n = 3 per group). (G) The expression of CD61 and CD41 in both BM MKs and spleen MKs was detected by flow cytometry after treatment for 12 days. (H) The histogram shows the percentage of CD41 + /CD61 + cells in BM MKs of each groups ( n = 3 per group). (I) The histogram shows the percentage of CD41 + /CD61 + cells in spleen MKs of each group ( n = 3 per group). Data represent the mean ± standard deviation (SD) of three independent experiments. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 vs. the model group. PE: phycoerythrin; FITC: fluorescein isothiocyanate; UL: upper left; UR: upper right; LL: lower left; LR: lower right.

Article Snippet: The cells were then treated with 8 μM fluorescein isothiocyanate (FITC)-conjugated anti-CD41 (Beijing 4A Biotech Co., Ltd., Beijing, China) and 8 μM phycoerythrin (PE)-conjugated anti-CD42b (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) in 100 μL of PBS.

Techniques: Staining, Control, Expressing, Flow Cytometry, Standard Deviation

Journal: Journal of Pharmaceutical Analysis

Article Title: Oxymatrine, a novel TLR2 agonist, promotes megakaryopoiesis and thrombopoiesis through the STING/NF-κB pathway

doi: 10.1016/j.jpha.2024.101054

Figure Lengend Snippet: The effect of megakaryocyte (MK) differentiation induced by oxymatrine was block by toll-like receptor 2 (TLR2) inhibitor C29 and stimulator of interferon genes (STING) inhibitor C-176. (A) Representative images of oxymatrine treated Meg-01 cells with oxymatrine (40 μM), C29 (50 μM), oxymatrine (40 μM) + C29 (50 μM), C-176 (10 μM), and oxymatrine (40 μM) + C-176 (10 μM) for five days. (B) The histogram represents the number of MKs in each group ( n = 3 per group). (C) Phalloidin-labeled cytospin in Meg-01 cells intervened with oxymatrine (40 μM), C29 (50 μM), oxymatrine (40 μM) + C29 (50 μM), C-176 (10 μM), and oxymatrine (40 μM) + C-176 (10 μM) for five days under a fluorescence Microscope. Excitation wavelength: 560 nm for phalloidin and 405 nm for 4′,6-diamidino-2-phenylindole (DAPI). (D) The histogram represents the relative mean fluorescence intensity (MFI) of F-actin in each group ( n = 3 per group). (E) Flow cytometry analysis exhibits the percentage of CD41 + /CD42 + cells after the treatment of oxymatrine (40 μM), C29 (50 μM), oxymatrine (40 μM) + C29 (50 μM), C-176 (10 μM), and oxymatrine (40 μM) + C-176 (10 μM) for five days. (F) The histogram represents the percentage of CD41 + /CD42b + expression in each group ( n = 3 per group). (G–J) Representative immunoblot images (G) and biochemical quantification of TLR2 (H), STING (I), and nuclear factor-kappaB (NF-κB) (J) after the treatment of oxymatrine (40 μM), C29 (50 μM), and oxymatrine (40 μM) + C29 (50 μM) for five days ( n = 3 per group). (K–N) Representative immunoblot images (K) and biochemical quantification of TLR2 (L), STING (M), and NF-κB (N) after the treatment of oxymatrine (40 μM), C-176 (10 μM), and oxymatrine (40 μM) + C-176 (10 μM) for five days ( n = 3 per group). Data represent the mean ± standard deviation (SD) of three independent experiments. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 vs. the oxymatrine group. ns: no significance. PE: phycoerythrin; FITC: fluorescein isothiocyanate; FOV: field of view; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: The cells were then treated with 8 μM fluorescein isothiocyanate (FITC)-conjugated anti-CD41 (Beijing 4A Biotech Co., Ltd., Beijing, China) and 8 μM phycoerythrin (PE)-conjugated anti-CD42b (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) in 100 μL of PBS.

Techniques: Blocking Assay, Labeling, Fluorescence, Microscopy, Flow Cytometry, Expressing, Western Blot, Standard Deviation