human pf4 platelet factor 4 elisa kit  (Elabscience Biotechnology)

Journal: EMBO Molecular Medicine

Article Title: 7D, a small molecule inhibits dengue infection by increasing interferons and neutralizing-antibodies via CXCL4:CXCR3:p38:IRF3 and Sirt1:STAT3 axes respectively

doi: 10.1038/s44321-024-00137-8

Figure Lengend Snippet: ( A , B ) Megakaryocytes isolated from mice bone marrow, and infected with DENV2 (MOI ~ 1) for 24 h with and without 7D. Culture supernatant was used for measuring ( A ) CXCL4 levels using ELISA, and ( B ) cell pallet was used for detecting viral genome using qRT PCR, n = 3 independent experiments for both ( P values: A : 0.0002; 0.04, B : 0.0002; 0.008). ( C , D ) Similar experiment was performed in monocytes (as CXCL4 non-producing cells) isolated from whole blood of these mice. ( C ) CXCL4 and ( D ) DENV2 genome were detected, n = 3 independent experiments for both assays ( P value: 0.0002). A similar experiment was performed in ( E , F ) human MEG-01 cell line (CXCL4 producing cells), n = 3 independent experiments ( P values: E : 0.0008; 0.01, F : 0.006) and ( G , H ) U937-DC-SIGN cell line (CXCL4 non-producing cells), n = 3 independent experiments for above assays. One-way ANOVA and Bonferroni’s post-test were used for data analysis. Data information: ( A – E , G ) One-way ANOVA and Bonferroni’s post-test were used for data analysis. Data are mean ± SEM. ( F , H ) Student’s t-test was used. Data are mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, ns=non-significant.

Article Snippet: Human PF4 ELISA (Elabscience, USA).

Techniques: Isolation, Infection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: EMBO Molecular Medicine

Article Title: 7D, a small molecule inhibits dengue infection by increasing interferons and neutralizing-antibodies via CXCL4:CXCR3:p38:IRF3 and Sirt1:STAT3 axes respectively

doi: 10.1038/s44321-024-00137-8

Figure Lengend Snippet: ( A ) Sirtuin-1 (Sirt1) expression in Sirt1 −/− and WT mouse embryonic fibroblasts (MEFs) in western blot assay. Densitometry analysis independent experiments n = 3, Student’s t test was used ( P value: 0.005). Above cells were infected with DENV2 and treated with 7D as mentioned for U937-DC-SIGN in Fig. . After experiment, ( B ) WT and ( C ) Sirt1 −/− cells were used for western blot analysis for NS1, Ac-P53 and P-IRF3. ( D – I ) Densitometry data from, n = 3 independent experiments, one-way ANOVA and Bonferroni’s post-test were used ( P values: D : 0.04; 0.007; 0.01, E : 0.001, F : 0.009; 0.0002; 0.009, G : 0.005; 0.0008; 0.0006, I : 0.006; 0.002; 0.007). ( J , K ) Viral genome was quantified in WT and Sirt1 −/− MEFs pellet using qRT-PCR, n = 3 independent experiments, one-way ANOVA and Bonferroni’s post-test were used ( P values: J : 0.0013; 0.0007; 0.0005; 0.006, K : 0.004; 0.0004; 0.002; 0.003). ( L , M ) IFNα level was measured using ELISA from cell supernatant of above experiments, n = 3 independent experiments, one-way ANOVA and Bonferroni’s post-test were used ( P values: L : 0.003; 0.01; 0.0001; 0.0001, M : 0.0001; 0.0002; 0.0001; 0.0001). Data information: ( A , D – M ) Data are mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns=non-significant.

Article Snippet: Human PF4 ELISA (Elabscience, USA).

Techniques: Expressing, Western Blot, Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: EMBO Molecular Medicine

Article Title: 7D, a small molecule inhibits dengue infection by increasing interferons and neutralizing-antibodies via CXCL4:CXCR3:p38:IRF3 and Sirt1:STAT3 axes respectively

doi: 10.1038/s44321-024-00137-8

Figure Lengend Snippet: ( A ) Schematic representation of mice experiment. AG129 mice were intraperitonially (i.p.) infected with 10 5 FFU mouse-adapted DENV2 virus (P8- P23085 INDI-60) or incomplete L15 media as mock. 7D (8 mg/kg body weight) was administered (i.p.) till 4 days post-infection (DPI) and other half were injected with vehicle (PBS + 10%Tween 80) and euthanized at 6 DPI for following assays. ( B ) In a similar experiment, mice were observed till 15 DPI to obtain survival curves using the Kaplan–Meier method. The curve comparison analysis was performed between the DENV2 ( n = 12) and DENV2 + 7D ( n = 14). Log-rank (Mantle Cox) test was used for statistical analysis ( P value: 0.03). ( C ) Change in the body weight was recorded till 15 DPI, n = 7 vehicle, n = 8 mice per group, two-way ANOVA was used for data analysis, ( P values: 0.009; 0.0001; 0.0001). ( D ) Plasma CXCL4 levels from above mice was measured using ELISA at 6 DPI, n = 5 vehicle, n = 8 mice per group, one-way ANOVA and Kruskal–Wallis test were used, ( P value: 0.005). ( E , F ) DENV2 genome was quantified by qRT-PCR in ( E ) liver and ( F ) spleen tissues, respectively, n = 6 vehicle, n = 8 mice per group, one-way ANOVA and Bonferroni’s post-test were used, ( P values: E : 0.001; 0.004, F : 0.001; 0.005). ( G – M ) ( G ) Platelet, ( H ) monocytes, ( I ) neutrophil and ( J ) platelet-leukocyte aggregates were measured from peripheral blood of mice from above experiment using flow cytometry, n = 5 vehicle, n = 8 mice per group, one-way ANOVA and ( G , H : Kruskal–Wallis test) and ( I , J : Bonferroni’s post-test) were used, ( P values: G : 0.0005; 0.03, H : 0.003; 0.03, J : 0.0001; 0.005). Gating strategy is mentioned in Appendix Fig. . Plasma levels of ( K ) IL6, ( L ) TNFα and ( M ) IL1β were measured using CBA assay, n = 3 vehicle, n = 6 mice per group, Mann–Whitney U test was used, ( P values: K : 0.02; L : 0.03; M : 0.004). ( N , O ) Vascular leakage in mice blood vessels. ( N ) Image of Evan’s blue dye extravasation from the veins in abdominal region of DENV2-infected mice. ( O ) Quantification of Evan’s blue dye in different tissues, n = 5 vehicle, n = 5 for DENV2 and, n = 6 for DENV2 + 7D, two-way ANOVA was used, ( P values: 0.02; 0.03; 0.04). ( P – R ) Plasma ( P ) IFNα, ( Q ) IFNβ, n = 4 vehicle, n = 8 mice per group, and ( R ) IFNγ levels, n = 5 vehicle, n = 6 mice per group, were measured using ELISA, one-way ANOVA and Bonferroni’s post-test were used, ( P values: P : 0.006; 0.006; Q : 0.01; R : 0.03). Data information: ( C – J , O – R ) Data are mean ± SEM, and ( K – M ) median ± IQR, * P < 0.05, ** P < 0.01, *** P < 0.001, ns non-significant. .

Article Snippet: Human PF4 ELISA (Elabscience, USA).

Techniques: Infection, Virus, Injection, Comparison, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Flow Cytometry, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: 7D, a small molecule inhibits dengue infection by increasing interferons and neutralizing-antibodies via CXCL4:CXCR3:p38:IRF3 and Sirt1:STAT3 axes respectively

doi: 10.1038/s44321-024-00137-8

Figure Lengend Snippet: ( A – D ) DENV2-specific antibodies ( A ) IgM at 3 DPI, and ( B ) IgG at 6 DPI were measured in mice plasma from the above experiments (Fig. ) using ELISA. A : n = 3 vehicle, n = 5 mice per group, one-way ANOVA and Bonferroni’s post-test, B : n = 4 vehicle, n = 8 DENV2, n = 9 DENV2 + 7D, Mann–Whitney U test was used, ( P values: A : 0.001; 0.03; B : 0.01). ( C ) FRNT 50 curves of neutralization activity of mice serum on DENV2 propagation in Vero cells, n = 3 vehicle, n = 10 mice per group. ( D ) Graph of the above values, n = 10 mice per group, Mann–Whitney U test was used ( P value: 0.02). Sera dilution assay is mentioned in Appendix Fig. . ( E ) Increased plasma cells (CD138 +ve B220 lo/-ve CD19 +ve ) percentage in 7D-treated mice spleen was measured using flow cytometry, n = 6 vehicle, n = 9 mice group, Kruskal–Wallis test is used ( P value: 0.02). ( F , G ) Spleenocytes from the above mice were used to quantitate DENV2-specific antibody secreting cells using ELISPOT assay, ( F ) representative image and ( G ) graph of the above values, n = 5 vehicle, n = 10 mice per group, one-way ANOVA and Bonferroni’s post-test were used ( P values: 0.0001; 0.04). ( H – J ) Intracellular levels of ( H , I ) Acetylated (Ac)-STAT3 and ( H , J ) phosphorylated ( P )-STAT3 in CD138 +ve cells was assessed by flow cytometry, n = 6 vehicle, n = 8 mice per group, one-way ANOVA and Bonferroni’s post-test were used ( P values: H : 0.0009; I : 0.001; 0.005; J : 0.001; 0.001). Gating strategy is mentioned in Appendix Fig. . ( K – M ) Western blot analysis of ( K , L ) Ac-STAT3 and ( K , M ) P-STAT3 from the spleenocytes normalized to β-actin. Densitometry of the above blots, n = 6 mice per group, one-way ANOVA and Bonferroni’s post-test were used ( P values: L : 0.001; M : 0.001). ( N , O ) Germinal center in spleen. ( N ) Immunofluorescent images of GL7-expressing follicles in spleen sections, GL7 (red) and DAPI (blue) staining. ( O ) Increased GL7-expressing follicles in 7D-treated mice, n = 8 mice per group, one-way ANOVA and Bonferroni’s post-test were used ( P values: 0.0001; 0.0002). Data information: ( A – C , E , G – J , L , M , O ) Data are mean ± SEM, and ( D ) median ± IQR, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns non-significant. Data from similar experiment in WT mice is described in Fig. . .

Article Snippet: Human PF4 ELISA (Elabscience, USA).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Neutralization, Activity Assay, Dilution Assay, Flow Cytometry, Enzyme-linked Immunospot, Western Blot, Expressing, Staining

Journal: EMBO Molecular Medicine

Article Title: 7D, a small molecule inhibits dengue infection by increasing interferons and neutralizing-antibodies via CXCL4:CXCR3:p38:IRF3 and Sirt1:STAT3 axes respectively

doi: 10.1038/s44321-024-00137-8

Figure Lengend Snippet: DENV2 infection experiment was performed in bone marrow derived macrophages (BMDMs), isolated from AG129 mice, in presence of 7D. A blocking antibody to IFNλ2/3 (5 µg/ml, standardization of working concentration is mentioned in Fig. EV4L below) was used to investigate the effects of type-III IFN. ( A ) Viral genome was quantified in cell pellets using qRT-PCR, n = 3 independent experiments ( P values: 0.0011; 0.0002; 0.03; 0.0014). ( B – E ) Relative gene expressions of interferon-stimulated genes (ISGs), ( B ) Oas1a, ( C ) Isg15, ( D ) Trim69, and ( E ) Mx1 were measured from above experiments, n = 3 independent experiments, ( P values: B : 0.0001; 0.0013; 0.0001; 0.0001, C : 0.0001; 0.0001; 0.0012; 0.01; 0.0001, D : 0.0006; 0.01; 0.0008; 0.0001, E : 0.0001; 0.0001; 0.0014; 0.009; 0.0001). ( F ) IFN λ2/3 levels were measured in the supernatant of these cells using ELISA, n = 3 independent experiments, ( P values: 0.004; 0.02; 0.002). ( G , H ) Relative gene expressions of ifnl2 and ifnl3 were quantified from the above cell pellets, n = 3 independent experiments, ( P values: G : 0.001; 0.001; 0.008, H : 0.001; 0.003; 0.001). ( I – K ) Western blot analysis for P-p38:p38 and P-IRF3:IRF3. ( J , K ) Densitometry data from, n = 3 independent experiments, ( P values: J : 0.007; 0.003; 0.001, K : 0.0006; 0.0002; 0.0003). ( L ) Concentration-dependent effect of blocking antibody against type-III IFN was tested on viral replication. DENV2 mRNA was quantified from pellets of DENV2-infected U937-DC-SIGN cells in presence of increasing concentration (1, 5 and 10 µg/ml) of blocking antibody against IFN-λ2/3 Ab, n = 3 independent experiments, ( P values: 0.001; 0.002; 0.0002). One-way ANOVA and Bonferroni’s post-test were used for above analysis. Data information: ( A – H , J – L ) One-way ANOVA and Bonferroni’s post-test were used. Data are mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 and ns=non-significant.

Article Snippet: Human PF4 ELISA (Elabscience, USA).

Techniques: Infection, Derivative Assay, Isolation, Blocking Assay, Concentration Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: EMBO Molecular Medicine

Article Title: 7D, a small molecule inhibits dengue infection by increasing interferons and neutralizing-antibodies via CXCL4:CXCR3:p38:IRF3 and Sirt1:STAT3 axes respectively

doi: 10.1038/s44321-024-00137-8

Figure Lengend Snippet: As mentioned in the above Fig. , a similar experiment was performed in CXCR3 −/− and WT C57BL/6 mice at 6 DPI. ( A , B ) Spleen was isolated and processed for measuring dengue viral genome copies were quantified in ( A ) CXCR3 −/− , n = 5 vehicle, n = 7 mice per group, one-way ANOVA and Kruskal–Wallis test, ( P values: 0.0002; 0.03), and ( B ) WT, n = 6 vehicle, n = 7 mice per group, one-way ANOVA and Bonferroni’s post-test, ( P values: 0.004; 0.04) using qRT-PCR. ( C , D ) IFNα levels were measured in plasma of these mice (C) CXCR3 −/− , n = 5 vehicle, n = 7 mice per group, and ( D ) WT, n = 6 vehicle, n = 9 mice per group, one-way ANOVA and Bonferroni’s post-test were used for both cases, ( P values: C : 0.0003, D : 0.01). ( E , F ) DENV2-specific IgG levels were measured in mice plasma of ( E ) CXCR3 −/− , n = 5 vehicle, n = 7 mice per group, and ( F ) WT, n = 5 vehicle, n = 6 mice per group, using ELISA. One-way ANOVA and Bonferroni’s post-test were used for both cases, ( P values: E : 0.003; 0.0013, F : 0.001). ( G – J ) FRNT 50 curves and graph of neutralization activity of mice plasma was measured in ( G , H ) CXCR3 −/− , n = 3 vehicle, n = 7 mice per group, and ( I , J ) WT, n = 6 vehicle, n = 7 mice per group. Mann–Whitney U test was used for analysis for both cases, ( P value: 0.003). Sera dilution assay is mentioned in Appendix Fig. . Data information: ( A – F , G , I ) Data are mean ± SEM, and ( H , J ) median ± IQR, * P < 0.05, ** P < 0.01, ns non-significant. .

Article Snippet: Human PF4 ELISA (Elabscience, USA).

Techniques: Isolation, Quantitative RT-PCR, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Neutralization, Activity Assay, MANN-WHITNEY, Dilution Assay

Journal: EMBO Molecular Medicine

Article Title: 7D, a small molecule inhibits dengue infection by increasing interferons and neutralizing-antibodies via CXCL4:CXCR3:p38:IRF3 and Sirt1:STAT3 axes respectively

doi: 10.1038/s44321-024-00137-8

Figure Lengend Snippet: As mentioned in the Fig. , a similar experiment was performed in AG129 mice treated with 7D and STAT3-inhibitor Stattic (10 mg/kg/body weight, referred concentration from vendor’s manual). ( A , B ) DENV2 viral RNA was quantified in liver and spleen respectively using qRT-PCR, n = 8 mice per group, ( P values: A : 0.0001; 0.0001; 0.003, B : 0.0001; 0.0003; 0.04). ( C , D ) Anti-dengue IgG was measured from the serum of mice using ELISA, n = 8, ( P values: 0.0001; 0.0005; 0.003). ( E – G ) Ac-STAT3 and P-STAT3 were measured in spleenocytes by western blot and normalized with total STAT3. Densitometry of the above blots, n = 4 vehicle, n = 6 mice per group ( P values: F : 0.0008; 0.0001, G : 0.01). One-way ANOVA and Bonferroni’s post-test were used for all above analysis. Data information: ( A – D , F , G ) One-way ANOVA and Bonferroni’s post-test were used. Data are mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns=non-significant.

Article Snippet: Human PF4 ELISA (Elabscience, USA).

Techniques: Concentration Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: EMBO Molecular Medicine

Article Title: 7D, a small molecule inhibits dengue infection by increasing interferons and neutralizing-antibodies via CXCL4:CXCR3:p38:IRF3 and Sirt1:STAT3 axes respectively

doi: 10.1038/s44321-024-00137-8

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Human PF4 ELISA (Elabscience, USA).

Techniques: Isolation, Sequencing, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Lysis, Plasmid Preparation, Software, Real-time Polymerase Chain Reaction

Journal: Heliyon

Article Title: The disparity of platelet factor 4 and platelets in individuals of different ages.

doi: 10.1016/j.heliyon.2024.e34923

Figure Lengend Snippet: Fig. 1. The expression of PF4 and CXCR3 varied in different age groups. (A) Young and Old peripheral blood, as well as Cord Blood samples, were processed and evaluated using ELISA, qPCR and FACS. (B) ELISA quantification of PF4 in PRP lysate of Cord Blood (n = 15), Young (23.40 ± 2.13 years, n = 15) and Old (75.23 ± 4.19 years, n = 13) peripheral blood samples. (C) Quantitative real-time PCR analysis of PF4 expression in Cord Blood (n = 15), Young (n = 15) and Old (n = 13) peripheral blood mononuclear cells. (D) Flow cytometry analysis of CXCR3 expression in mononuclear cells from Cord Blood (n = 15), Young (n = 15) and Old (n = 13) peripheral blood. Data are presented as mean ± SD and analyzed using One-way ANOVA. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: The PF4 level in PRP lysate was measured using a Human Platelet Factor, PF4 ELISA kit (CUSABIO, CSB-E07882h) according to the manufacturer’s protocol.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Flow Cytometry

Journal: Heliyon

Article Title: Platelet morphology, ultrastructure and function changes in acute ischemic stroke patients based on structured illumination microscopy

doi: 10.1016/j.heliyon.2023.e18543

Figure Lengend Snippet: PF4 content was lower in AIS patient but release more after being stimulated while 2MeSAMP inhibited PF4 releasing. A, PF4 content of elder healthy in 7 different groups (n = 3). PF4 release increased after 5 min and 1 h of thrombin stimulation (****P < 0.0001; ****P < 0.0001). Releasing of PF4 decreased after dealing with 2MeSAMP for 5 min and 1 h (***P = 0.0001). B , PF4 content of AIS patients in 7 different groups (n = 3). PF4 release increased after 5 min and 1 h of thrombin stimulation (****P < 0.0001; ****P < 0.0001). Releasing of PF4 decreased after dealing with 2MeSAMP for 5 min and 1 h (**P = 0.0062; **P = 0.0019). C , PF4 content was lower in AIS patients (n = 3) at rest stage (****P < 0.0001). D , Ratio of PF4 content after activation to before. Stimulated with 0.5U TH for 5 min, the ratio was higher in AIS patients (n = 3) compared with EH (n = 3) (***P = 0.0007). E , Ratio of PF4 content after drug administration to before. The ratio was lower after treatment with 2MeSAMP for 5 min and 1 h compared with aspirin (**P = 0.0012; **P = 0.003). Unpaired two-tailed Student's t-test. Data are presented as Mean value standard deviation.

Article Snippet: Assays were performed in accordance with instructions of Human PF4 ELISA kit (Cusabio, Wuhan, China, lot: CSB-E07882).

Techniques: Activation Assay, Two Tailed Test, Standard Deviation