Journal: Journal of Extracellular Vesicles

Article Title: Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium

doi: 10.1002/jev2.12044

Figure Lengend Snippet: Purity of EV samples assessed by particle/protein ratio and Western blotting. (a)‐(c) The protein concentrations of EV preparations separated by different methods from CCM, urine and plasma. The protein concentrations have been corrected for initial sample input volume. (d)‐(f) The particle/protein ratios of EV preparations separated by different methods from CCM, urine and plasma. The particle numbers were measured by nFCM. The error bars represented the standard deviation of three repetitive experiments. * P < 0.05, ** P < 0.01, one‐way ANOVA analysis with Tukey multiple comparison test as well as a variance‐covariance model. (g) Western blots of flotillin‐1, CD63, CD81, and calnexin for EV preparations separated from CCM by different methods. 9 μg of protein from each EV preparation was loaded. (h) Western blots of flotillin‐1, CD63, CD81, THP and calnexin for EV preparations separated from urine by different methods. 3.3 μg of protein from each EV preparation was loaded. (i) Western blots of flotillin‐1, CD81, APOA1 and calnexin for EV preparations separated from plasma by different methods. 25 μg of protein from each EV preparation was loaded. MCF7 membrane and cytosolic protein fractions served as positive and negative controls for flotillin‐1, CD63, CD81 and calnexin. Whole urine sample was used as positive control and MCF7 cellular fractions as negative control for THP. Recombinant human Apolipoprotein A1 served as positive control for APOA1.GAPDH staining was to show proper loading and running of the cytosolic MCF7 fraction

Article Snippet: Overnight incubation at 4°C of the membranes was performed with the following primary antibodies: THP (1:1,000, mouse monoclonal B‐2, #sc‐271022 Santa Cruz Biotechnology); and APOA1 (1:5,000, goat polyclonal, #ab7613, Abcam).

Techniques: Western Blot, Clinical Proteomics, Standard Deviation, Comparison, Membrane, Positive Control, Negative Control, Recombinant, Staining

Journal: Blood Advances

Article Title: Chimeric antigen receptor–T cells with cytokine neutralizing capacity

doi: 10.1182/bloodadvances.2019001287

Figure Lengend Snippet: Functionality of mbaIL6. (A) Jurkat cells (2 × 106/mL) transduced with either GFP alone (“Control”) or GFP plus mbaIL6 were cultured for 2 hours with 1 ng/mL human IL-6; IL-6 in the supernatant was measured by ELISA. Mean (±SD; n = 3) is shown. ****P < .0001. (B) Cultures were set up as in panel A; IL-6 levels were measured after the indicated time. The dashed curve is the fitted exponential decay curve. Mean (±SD; n = 3) is shown. (C) Cultures were set as in panel A but with various Jurkat cell concentrations; IL-6 was measured after 2 hours. Mean (±SD; n = 3) for each cell concentration is shown. **P < .01; ***P < .001; ****P < .0001. (D) Cultures were initiated with 0.1 × 106 cells/mL Jurkat cells; cell numbers increased to 0.2 × 106/mL after 24 hours, 0.4 × 106/mL after 48 hours, and 1.0 × 106/mL after 72 hours. Mean (±SD) for each time point is shown (n = 3). **P < .01; ****P < .0001. (E) Jurkat cells (2 × 106/mL) nontransduced (“wt”) or transduced with mbaIL6 were cultured for 2 hours with 1 ng/mL IL-6. U937 cells were exposed to the supernatant for 15 minutes at 37°C. Representative flow cytometry histograms show labeling of U937 cells with anti-STAT3 pY705. (F) Mean (±SD; n = 3) STAT3 phosphorylation in U937 cells. ***P < .001. (G) Varying concentrations of Jurkat cells transduced with mbaIL6 were cultured with 1 ng/mL IL-6 for 2 hours. The supernatant was added to 0.2 × 106 DS-1 cells, which were counted after 7 and 9 days of culture. Mean (±SD; n = 3) is shown. (H) Jurkat cells expressing mbaIL6 were cultured with IL-6 (5 ng/mL) for 2 hours; after washing, cells were cultured for another 2 hours and periodically labeled with anti–IL-6 PE. Flow cytometry dot plots show levels of IL-6 bound to mbaIL6 cells. Sequential data of 2 experiments are given in supplemental Figure 2C. (I) Jurkat cells (1 × 106/mL) expressing mbaIL6 were cultured with IL-6 (5 ng/mL) for 2 hours; after washing, cells were cultured for another 48 hours and periodically labeled with anti–IL-6 PE. Graph shows mean fluorescence intensity (MFI) of IL-6 (red) plotted together with cell count (blue). Mean (±SD; n = 3) is shown. (J) Supernatant from cultures shown in panel I was added to THP-1 cells for 15 minutes at 37°C. STAT3 phosphorylation was measured as in panel E. HL, half-life.

Article Snippet: In some experiments, THP-1 cells were exposed to 0 to 10 µg/mL tocilizumab (Selleck Chemicals, Houston, TX) for 30 minutes before IL-6 stimulation.

Techniques: Transduction, Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay, Flow Cytometry, Labeling, Expressing, Fluorescence, Cell Counting

Journal: Blood Advances

Article Title: Chimeric antigen receptor–T cells with cytokine neutralizing capacity

doi: 10.1182/bloodadvances.2019001287

Figure Lengend Snippet: Functional consequences of IL-6 neutralization with mbaIL6-T cells. (A) mbaIL6 and GFP expression in peripheral blood T cells transduced with either GFP alone (“Control”) or GFP plus mbaIL6, after labeling with biotin-conjugated goat anti-human F(ab′)2 antibody and streptavidin-APC. (B) IL-6 binding to Control or mbaIL6-transduced peripheral blood T cells, labeled with IL-6 biotin and streptavidin-APC. (C) Cell marker profile of Control or mbaIL6-transduced T cells from 3 donors. Mean (±SD) of percent T cells expressing each marker is shown. (D) Control or mbaIL6-transduced T lymphocytes at the indicated concentrations were cultured for 2 hours with 1 ng/mL human IL-6; IL-6 in the supernatant was measured by using ELISA. Mean (±SD; n = 3) is shown. ***P < .001. (E) DS-1-mCherry cells were cocultured with Control or mbaIL6-transduced T cells at a 1:1 ratio, with IL-6 (0.5 ng/mL). DS-1 proliferation was quantitated by using the IncuCyte Live Imaging System; shown are mean (±SD) of red calibrated units (RCU) × μm2/well in triplicate measurements. *P = .02; **P < .01 for data at 120 hours. (F) Control or mbaIL6-transduced T lymphocytes at the indicated concentration were cultured for 2 hours with 1 ng/mL IL-6. THP-1 cells were then exposed to either 1 ng/mL IL-6 or to the supernatant of the lymphocyte cultures for 15 minutes at 37°C. Flow cytometry histograms show labeling of THP-1 cells with anti-STAT3 pY705; the graph on the right shows the decrease in pSTAT3 relative to that of THP-1 cells exposed for 15 minutes to 1 ng/mL IL-6. (G) THP-1 cells were exposed to IL-6 for 15 minutes, after 30-minute incubation with the indicated concentrations of tocilizumab. Cells were then labeled with anti-STAT3 pY705 and analyzed as in panel F.

Article Snippet: In some experiments, THP-1 cells were exposed to 0 to 10 µg/mL tocilizumab (Selleck Chemicals, Houston, TX) for 30 minutes before IL-6 stimulation.

Techniques: Functional Assay, Neutralization, Expressing, Transduction, Labeling, Binding Assay, Marker, Cell Culture, Enzyme-linked Immunosorbent Assay, Imaging, Concentration Assay, Flow Cytometry, Incubation

Journal: Blood Advances

Article Title: Chimeric antigen receptor–T cells with cytokine neutralizing capacity

doi: 10.1182/bloodadvances.2019001287

Figure Lengend Snippet: Function of T cells expressing mbaIL6 and anti-CD19 CAR cocultured with a monocytic cell line. (A) Cytotoxicity of T cells against OP-1-mCherry cells, with or without undifferentiated THP-1 cells (1:5:1 T-cell:OP-1:THP-1 ratio). OP-1 growth was quantitated by using the IncuCyte Live Imaging System; results are expressed as mean (±SD) of red calibrated units (RCU) × μm2/well in triplicate measurements. (B) IL-6 levels in the supernatant of the cultures shown in panel A after 48 hours, measured by using ELISA. CAR expression was 84% to 97% for CAR alone and 86% to 96% for CAR plus mbaIL6 (“DUAL”). Mean (±SD) of 3 measurements each with T cells from 2 donors. Not significant (n.s.), P > .05; ****P < .0001. (C) Supernatant from CAR-T/OP-1 48-hour cocultures was added to T cells mixed at 1:1 with either undifferentiated or differentiated THP-1 cells (supplemental Figure 6D). Cytokine levels were measured after an additional 72 hours of culture. Results with T cells transduced with GFP only or mbaIL6 are shown in supplemental Figure 6E.

Article Snippet: In some experiments, THP-1 cells were exposed to 0 to 10 µg/mL tocilizumab (Selleck Chemicals, Houston, TX) for 30 minutes before IL-6 stimulation.

Techniques: Expressing, Imaging, Enzyme-linked Immunosorbent Assay, Transduction

Journal: Blood Advances

Article Title: Chimeric antigen receptor–T cells with cytokine neutralizing capacity

doi: 10.1182/bloodadvances.2019001287

Figure Lengend Snippet: CAR–T cells expressing mbaIL6 quench IL-6 and exert antileukemia activity in xenograft models. (A) NOD/scid-IL2RGnull mice were injected IV with 0.5 to 1 × 106 Nalm-6-luciferase cells. On day 3, mice were given T cells expressing either anti-CD19 CAR alone (85% CAR expression) or mbaIL6 plus CAR (“DUAL”; 79% CAR expression) (20 × 106/mouse IV); all mice received 20 000 IU IL-2 IP every 2 days. Ventral images from the Xenogen IVIS-200 system after D-luciferin injection are shown (captured with enhanced sensitivity on day 3 to visualize Nalm-6 engraftment; full set of ventral and dorsal images is shown in supplemental Figure 7). (B) Luminescence measurements (photons per second) in the mice. Each point corresponds to a measurement in 1 mouse. (C) Levels of GFP+ CD3+ CAR–T cells in blood 50 days after CAR–T cell injection in a subset of the mice. (D) Kaplan-Meier curves of overall survival for the mice shown in panel A, euthanized when the total bioluminescence signal reached 1 × 1010 photons/second. **P < .01 by log-rank test. (E) T cells expressing either anti–CD19 CAR or anti–CD19 CAR plus mbaIL6 were injected IV in NOD/scid-IL2RGnull mice (2-10 × 106/mouse); 3 days later, 50 ng of human IL-6 was injected IP. After 2 hours, mice were euthanized, and serum was collected by cardiac puncture to measure levels of human IL-6 by using ELISA. Each symbol corresponds to data from 1 mouse; bars show mean (±SD). **P < .01. (F) Mice from the experiments shown in panel E were divided according to the number of T cells that were administered: 2 to 4 × 106 (“low”) and 5 to 10 × 106 (“high”). Values correspond to the percentage of IL-6 that was removed from serum in each mouse, using as a reference the mean value of IL-6 measured in mice that received IL-6 with no prior injection of T cells. *P = .035 for CAR low vs DUAL low, P = .045 from CAR high vs, DUAL low, P = .013 for DUAL low vs DUAL high; ***P < .001. (G) Daudi-luciferase cells were injected IP in NOD/scid-IL2RGnull mice (20 × 106/mouse), followed 3 days later by THP-1 and/or T cells IP (20 × 106 for both cell types). Tumor engraftment was measured by in vivo imaging (supplemental Figure 8). Mice were euthanized 48 hours after THP-1 and/or T-cell injection. Symbols show IL-6 levels measured by using ELISA in peritoneal lavage, according to percentage of tumor reduction. *P = .032 for CAR vs no T cells; P = .046 for CAR vs DUAL. (H) IL-6 binding to T cells from the peritoneal lavage of 4 mice, 2 injected with CAR–T cells and 2 injected with T cells expressing both CAR and mbaIL6. Cells were stained with anti-mouse CD45-PE-Cy7, anti-human CD45-PerCP, anti-human CD3-APC, and anti-human IL-6-PE; the plots show selectively gated mouse CD45–, human CD45+, and human CD3+ cells.

Article Snippet: In some experiments, THP-1 cells were exposed to 0 to 10 µg/mL tocilizumab (Selleck Chemicals, Houston, TX) for 30 minutes before IL-6 stimulation.

Techniques: Expressing, Activity Assay, Injection, Luciferase, Enzyme-linked Immunosorbent Assay, In Vivo Imaging, Binding Assay, Staining

Journal: Cell Reports Medicine

Article Title: Macrophages are activated toward phagocytic lymphoma cell clearance by pentose phosphate pathway inhibition

doi: 10.1016/j.xcrm.2024.101830

Figure Lengend Snippet: Cross validation of PPP inhibition in macrophages confirms increased ADCP rates (A–C) ADCP change compared to basal phagocytosis rate of J774A.1 macrophages under inhibition of PPP. (A) Alternative inhibitor physcion of 6-phosphogluconate dehydrogenase (6Pgd) in oxidative part of PPP (red) and p -hydroxyphenylpyruvate for inhibition of transketolase (Tkt) in non-oxidative part of PPP (blue). (B) Using human monocyte cell line THP1 and CD20 antibody obinutuzumab under inhibition of 6Pgd by 6-aminonicotinamide (red) and inhibition of Tkt by oxythiamine (blue). (C) ADCP assay performed under hypoxic conditions and inhibition of 6Pgd by physcion (red) or inhibition of Tkt by oxythiamine (blue). (D) Antibody-independent cellular phagocytosis of hMB cells by J774A.1 macrophages compared to control under inhibition of 6Pgd by 6-aminonicotinamide (left) and physcion (right). (E) ADCP change compared to basal phagocytosis rate of empty vector control J774A.1 macrophages under shRNA-mediated knockdown of 6Pgd (red) and Tkt (blue). (F) ADCP change compared to basal phagocytosis rate of J774A.1 macrophages under supplementation of metabolites of the PPP. Enzyme reactions in focus colored in violet (6Pgd) and blue (Tkt). E4P , erythrose-4-phosphate; F6P , fructose-6-phosphate; G3P , glyceraldehyde-3-phosphate; Glc6P , glucose-6-phosphate; R5P , ribose-5-phosphate; Ru5P , ribulose-5-phosphate; S7P , sedoheptulose-7-phosphate; X5P , xylulose-5-phosphate. Data are shown as mean ± SEM. Technical replicates (A) n = 30, (B) n = 17–25, (C) n = 25–28, (D) n = 30, (E) n = 20–23, (F) n = 13–20; biological replicates (A) n = 6, (B) n = 4–5, (C) n = 5–6, (D) n = 6, (E) n = 4–5, (F) n = 3–4. p values were calculated using one-way ANOVA. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. See also Figures S1 and .

Article Snippet: HEK293T-CAF40-null cells were from DSMZ, hMB cells were generated by Leskov et al., J774A.1 macrophages were from ATCC, THP1 monocytes were from DSMZ. hMB cells represent a humanized mouse model of “double-hit” lymphoma by overexpression of c-MYC and BCL2 in human HSC-derived B-lineage cells. hMB cells express GFP and can be targeted by antibodies used in clinics.

Techniques: Biomarker Discovery, Inhibition, Control, Plasmid Preparation, shRNA, Knockdown

Journal: Biomedicines

Article Title: Human Regulatory Macrophages Derived from THP-1 Cells Using Arginylglycylaspartic Acid and Vitamin D3

doi: 10.3390/biomedicines11061740

Figure Lengend Snippet: Schema of procedures to generate different types of macrophages using human THP-1 monocytes.

Article Snippet: The human monocytic cell line, THP-1 (Korean Cell Line Bank, Seoul, Republic of Korea) was grown in RPMI-1640 medium supplemented with 5% heat-inactivated fetal bovine serum (FBS) and 1% antibiotic-antimycotic.

Techniques:

Journal: Biomedicines

Article Title: Human Regulatory Macrophages Derived from THP-1 Cells Using Arginylglycylaspartic Acid and Vitamin D3

doi: 10.3390/biomedicines11061740

Figure Lengend Snippet: Incubation with PMA, RGD, and vitD3 induces THP-1 cell elongation. We incubated THP-1 cells with PMA (10 ng/mL) for 2 days, followed by various concentrations of RGD ( A ) or vitD3 ( B ) for 1 day. Cells were incubated with PMA (10 ng/mL) for 2 days, followed by RGD (1 μg/mL), then 1 μM vitD3 for 1 day (PMA + RGD + VitD3 group; ( C ) Cell morphology was visualized using an inverted microscope at 100× magnification.

Article Snippet: The human monocytic cell line, THP-1 (Korean Cell Line Bank, Seoul, Republic of Korea) was grown in RPMI-1640 medium supplemented with 5% heat-inactivated fetal bovine serum (FBS) and 1% antibiotic-antimycotic.

Techniques: Incubation, Inverted Microscopy

Journal: Biomedicines

Article Title: Human Regulatory Macrophages Derived from THP-1 Cells Using Arginylglycylaspartic Acid and Vitamin D3

doi: 10.3390/biomedicines11061740

Figure Lengend Snippet: Incubation with PMA, RGD, and vitD3 upregulated IL-10 and downregulated IL-12 in THP-1 cells. We incubated THP-1 cells with 10 ng/mL PMA for 2 days, washed them with DPBS, then maintained them in RPMI-1640 media for 2 days (PMA group). Cells were incubated with PMA (10 ng/mL) for 2 days, followed by RGD (1 μg/mL; PMA + RGD) or vitD3 (1 μM; PMA + VitD3) for 1 day. Cells were washed with DPBS, then maintained in RPMI-1640 media for 1 day. We incubated cells with PMA (10 ng/mL) for 2 days, followed (sequentially) by RGD (1 μg/mL) for 1 day and vitD3 (1 μM) for 1 day (PMA + RGD + VitD3). Cells were harvested, and the mRNA expressions of anti-inflammatory IL-10 ( A ) and pro-inflammatory IL-12 ( B ) were quantified using qRT-PCR. * p < 0.05, ** p < 0.005 vs. controls incubated with DMSO; # p < 0.05, ## p < 0.005.

Article Snippet: The human monocytic cell line, THP-1 (Korean Cell Line Bank, Seoul, Republic of Korea) was grown in RPMI-1640 medium supplemented with 5% heat-inactivated fetal bovine serum (FBS) and 1% antibiotic-antimycotic.

Techniques: Incubation, Quantitative RT-PCR

Journal: Biomedicines

Article Title: Human Regulatory Macrophages Derived from THP-1 Cells Using Arginylglycylaspartic Acid and Vitamin D3

doi: 10.3390/biomedicines11061740

Figure Lengend Snippet: Phenotypic characterization of PMA/RGD/vitD3-induced Mregs-like cells. We generated resting macrophages by incubating THP-1 cells with PMA (10 ng/mL) for 2 days, washing them with DPBS, then maintaining them in RPMI-1640 media for 2 days (resting macrophages group). Cells were incubated with PMA (10 ng/mL) for 2 days, followed by RGD (1 μg/mL) for 1 day, then with vitD3 (1 μM) for 1 day to generate Mregs. Cells were harvested, and the expression of the cell surface and intracellular markers were assessed using flow cytometry. ( A ) Black lines, red lines, and gray shadows on representative flow cytometric histograms indicate resting macrophages, Mregs, and controls, respectively. ( B ) Bar graphs show the mean values of relative protein expression ± SD. * p < 0.05, ** p < 0.005 vs. DMSO controls. # p < 0.05, ## p < 0.005.

Article Snippet: The human monocytic cell line, THP-1 (Korean Cell Line Bank, Seoul, Republic of Korea) was grown in RPMI-1640 medium supplemented with 5% heat-inactivated fetal bovine serum (FBS) and 1% antibiotic-antimycotic.

Techniques: Generated, Incubation, Expressing, Flow Cytometry

Journal: Biomedicines

Article Title: Human Regulatory Macrophages Derived from THP-1 Cells Using Arginylglycylaspartic Acid and Vitamin D3

doi: 10.3390/biomedicines11061740

Figure Lengend Snippet: Marker expression of Mregs did not change upon exposure to LPS. ( A – D ) We generated Mregs as described in . We stimulated THP-1 cells or Mregs with LPS (100 ng/mL) for 24 h, then quantified expression of cell surface markers using flow cytometry. For ( A , C ), the red and black lines are representative of the flow cytometric histograms for Mregs and Mregs + LPS, respectively. Pink lines and gray shadow indicate THP-1 + LPS and controls, respectively. For ( B , D ), Bar graphs show the mean values of relative protein expression ± SD. * p < 0.05, ** p < 0.005 vs. controls incubated with DMSO; ## p < 0.005.

Article Snippet: The human monocytic cell line, THP-1 (Korean Cell Line Bank, Seoul, Republic of Korea) was grown in RPMI-1640 medium supplemented with 5% heat-inactivated fetal bovine serum (FBS) and 1% antibiotic-antimycotic.

Techniques: Marker, Expressing, Generated, Flow Cytometry, Incubation

Journal: Biomedicines

Article Title: Human Regulatory Macrophages Derived from THP-1 Cells Using Arginylglycylaspartic Acid and Vitamin D3

doi: 10.3390/biomedicines11061740

Figure Lengend Snippet: Marker expression of Mregs did not change upon exposure to xenogeneic antigen. ( A – D ) We generated Mregs as described in . We stimulated THP-1 cells or Mregs with α-gal (100 ng/mL) for 24 h, then quantified the expression of cell surface markers using flow cytometry. ( A , C ) are the representative flow cytometric histograms, for which red and black lines indicate Mregs and Mregs + α-gal, respectively, while pink lines and gray shadow indicate THP-1 + α-gal and controls, respectively. ( B , D ) are the mean values of relative protein expression ± SD. * p < 0.05, ** p < 0.005 vs. controls incubated with DMSO; ## p < 0.005.

Article Snippet: The human monocytic cell line, THP-1 (Korean Cell Line Bank, Seoul, Republic of Korea) was grown in RPMI-1640 medium supplemented with 5% heat-inactivated fetal bovine serum (FBS) and 1% antibiotic-antimycotic.

Techniques: Marker, Expressing, Generated, Flow Cytometry, Incubation

Journal: Biomedicines

Article Title: Human Regulatory Macrophages Derived from THP-1 Cells Using Arginylglycylaspartic Acid and Vitamin D3

doi: 10.3390/biomedicines11061740

Figure Lengend Snippet: Regulatory macrophages expressed fewer inflammatory and coagulation-related genes and more immunosuppressive genes in response to pig endothelial cell line MPN3. We generated M1 macrophages by incubating THP-1 cells with LPS (100 ng/mL) and IFN-γ (20 ng/mL) for 24 h, and generated Mregs as described in . Thereafter, MPN-3 cells were cocultured with THP-1, M1, or Mregs, for 2 h (fgl-2, and TF), 6 h (IDO, IL-1β, IL-6, IL-10, IL-12, iNOS, and TNF-α), or 12 h (PAR-1 and MCP-1), respectively. Cells were harvested, and the mRNA expression of cytokines and chemokines ( A ), as well as coagulation-related genes ( B ), were evaluated using qRT-PCR. * p < 0.05, ** p < 0.005 vs. MPN3 + THP-1; ## p < 0.005.

Article Snippet: The human monocytic cell line, THP-1 (Korean Cell Line Bank, Seoul, Republic of Korea) was grown in RPMI-1640 medium supplemented with 5% heat-inactivated fetal bovine serum (FBS) and 1% antibiotic-antimycotic.

Techniques: Coagulation, Generated, Expressing, Quantitative RT-PCR

Journal: Biomedicines

Article Title: Human Regulatory Macrophages Derived from THP-1 Cells Using Arginylglycylaspartic Acid and Vitamin D3

doi: 10.3390/biomedicines11061740

Figure Lengend Snippet: Regulatory macrophages suppressed mRNA expression of pro-inflammatory and coagulation-related genes in M1 and MPN3 cocultures, according to the ratios of Mregs and M1 cells. We generated M1 macrophages by incubating THP-1 cells with LPS (100 ng/mL) and IFN-γ (20 ng/mL) for 24 h, in order to generate Mregs as described in . Thereafter, Mregs were cocultured them at indicated ratios for 1 day. Thereafter, THP-1, M1, or Mregs/M1 cocultures were incubated with MPN-3 cells for 2 h (fgl-2, and TF), 6 h (IDO, IL-1β, IL-6, IL-10, IL-12, iNOS, and TNF-α), or 12 h (PAR-1 and MCP-1). Cells were harvested and mRNA expression of cytokines and chemokines ( A ) and coagulation-related genes ( B ) were evaluated using qRT-PCR. * p < 0.05, ** p < 0.005 vs. MPN3 + THP-1; # p < 0.05, ## p < 0.005.

Article Snippet: The human monocytic cell line, THP-1 (Korean Cell Line Bank, Seoul, Republic of Korea) was grown in RPMI-1640 medium supplemented with 5% heat-inactivated fetal bovine serum (FBS) and 1% antibiotic-antimycotic.

Techniques: Expressing, Coagulation, Generated, Incubation, Quantitative RT-PCR