Journal: Clinica chimica acta; international journal of clinical chemistry

Article Title: Total testosterone quantitative measurement in serum by LC-MS/MS ☆

doi: 10.1016/j.cca.2014.06.009

Figure Lengend Snippet: Accuracy traceability to JCTLM listed materials and NIST serum reference materials.

Article Snippet: Serum-based reference material SRM 971 at 2 concentrations was obtained from NIST, and ERM-DA345 and ERM-DA346 from LGC Standards for accuracy assessment.

Techniques:

Journal: PLoS ONE

Article Title: The In Vitro Effect of Acidic-Pepsin on Nuclear Factor KappaB Activation and Its Related Oncogenic Effect on Normal Human Hypopharyngeal Cells

doi: 10.1371/journal.pone.0168269

Figure Lengend Snippet: Luciferase assay demonstrates the effect of pepsin in NF-κB transcriptional activity in (A) HHK and (B) HHPC. Acidic-pepsin (pH 4.0) does not induce NF-κB transcriptional activity in treated human normal hypopharyngeal cells. Columns represent NF-κB relative transcriptional activity (NF-κB responsive luciferase reporter/control luciferase reporter). 3kB-ConA-luc: NF-κB responsive luciferase reporter; ConA-luc: control luciferase reporter; 3kB-ConA-luc/ConA-luc: NF-κB responsive luciferase reporter/control luciferase reporter. CA: Acid-Control; PA: Acidic-Pepsin; CW: Weakly Acidic-Control; PW: Weakly Acidic-Pepsin; CN: Neutral-Control; PN: Neutral-Pepsin; PI: Inactivated-Pepsin.

Article Snippet: The HHPC were plated in non-coated flasks and were grown in Human Hypopharyngeal Normal Cell Culture Media with Serum (Celprogen Inc. CA, USA), at 37°C in humidified air and 5% CO 2 .

Techniques: Luciferase, Activity Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Bacterial lipoproteins constitute the TLR2-stimulating activity of Serum Amyloid A

doi: 10.4049/jimmunol.1800503

Figure Lengend Snippet: Recombinant SAA produced in E. coli, but not produced from eukaryotic cells, stimulates pro-inflammatory cytokine production. Primary human PBMCs (A-D) and neutrophils (PMNs) (E) were unstimulated (control) or stimulated with 62, 250, or 1000 ng/ml of SAA from different sources for 24 (PBMC) or 3 hours (PMN), after which cytokine concentrations were measured by ELISA. Primary C57BL/6J mouse splenocytes (F), mouse RAW macrophage cells (G), and J774 mouse macrophages (H) were unstimulated (control) or stimulated with 1¼g/ml of SAA from different sources. Culture supernatants were collected 24 hours later and TNFα concentrations were measured by ELISA. J774 mouse macrophages were unstimulated (control) or stimulated for 24 hours with 100 ng/ml of LPS, 1 ¼g/ml of apoSAA, LPS + 100 ng/ml of the lipopeptide Pam3CSK4 (Pam), LPS + apoSAA, or LPS + 1 ¼g/ml of human SAA1 made in HEK cells, and IL-1β concentrations in culture supernatants were measured by ELISA (I). J774 mouse macrophages were unstimulated (control) or stimulated for 24 hours with 10, 100, or 1000 ng/ml of SAA from different sources and TNFα concentrations in culture supernatants were measured 24 hours later by ELISA (J). 250 ¼g of apoSAA was separated by FPLC using a size exclusion column and 90 fractions were collected (K). Fractions obtained from FPLC were diluted 1:10 in serum-free media and used to stimulate J774 cells for 24 hours after which TNFα concentrations were measured by ELISA (L). Data are mean ± SEM and are representative of three independent experiments with n = 4 per group. Statistics were analyzed using one-way ANOVA with Dunnett’s test for multiple comparisons. **** p < 0.0001, ** p < 0.01, * p < 0.05.

Article Snippet: Human SAA1 from Origene produced in HEK cells (hSAA1) or ovalbumin (Ova) was unexposed or exposed to Pam3CSK4 (Pam) overnight (control), and some of the preparation was subjected to concentration of proteins >3 kDa using concentrated using Amicon Ultra centrifugal concentrators (filtered).

Techniques: Recombinant, Produced, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Bacterial lipoproteins constitute the TLR2-stimulating activity of Serum Amyloid A

doi: 10.4049/jimmunol.1800503

Figure Lengend Snippet: apoSAA, but not SAA1 produced from eukaryotic cells, induces TNFα secretion from macrophages and promotes IL-17A production from CD4 T cells. HEK cells were transiently transfected with empty vector (pcDNA3) or plasmids encoding human SAA1 (hSAA1.pcDNA3 or hSAA1.pCMV6XL5). 48 hours later, culture supernatants were collected and concentrated using Amicon Ultra centrifugal concentrators, SAA1 concentrations were measured by ELISA (A), and the culture supernatants were used to stimulate J774 cells for 24 hours (in the absence or presence of 10 ng/ml apoSAA) after which TNFα production was measured by ELISA (B). Similarly-prepared HEK supernatants were added to BMDCs for 48 hours, their culture supernatants were collected and added to CD4+ T-cells that were stimulated with 5 ¼g/ml immobilized anti-CD3 and 2 ¼g/ml soluble anti-CD28 for 72 hours after which IL-4 (C), IFNγ (D), and IL-17A (E) were measured by ELISA. Data are mean ± SEM and are representative of three (A-B) or two (C-E) independent experiments with n = 4 per group. Statistics were analyzed using one-way ANOVA with Dunnett’s (A-B) or Tukey’s (C-E) test for multiple comparisons. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Human SAA1 from Origene produced in HEK cells (hSAA1) or ovalbumin (Ova) was unexposed or exposed to Pam3CSK4 (Pam) overnight (control), and some of the preparation was subjected to concentration of proteins >3 kDa using concentrated using Amicon Ultra centrifugal concentrators (filtered).

Techniques: Produced, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Bacterial lipoproteins constitute the TLR2-stimulating activity of Serum Amyloid A

doi: 10.4049/jimmunol.1800503

Figure Lengend Snippet: Lipopeptides associated with SAA confer its ability to stimulate TNFα production. 100 ng/ml LPS (A), 100 ng/ml Pam3CSK4 (Pam, B), and 1¼g/ml apoSAA (C) were incubated with lipoprotein lipase (LPL), and then used to stimulate J774 cells. Culture supernatants were collected 24 hours later and TNFα concentrations were measured by ELISA. Human SAA1 from Origene produced in HEK cells (hSAA1) or ovalbumin (Ova) was unexposed or exposed to Pam3CSK4 (Pam) overnight (control), and some of the preparation was subjected to concentration of proteins >3 kDa using concentrated using Amicon Ultra centrifugal concentrators (filtered). Control and filtered preparations were used to stimulate J774 cells for 24 hours after which TNFα concentrations in culture media were measured by ELISA (D). Data are mean ± SEM and are representative of three (A-C) or two (D) independent experiments with n = 4 per group. Statistics were analyzed using one-way ANOVA with Dunnett’s test for multiple comparisons (A-C) or two-way ANOVA with Tukey’s test for multiple comparisons (D). **** p < 0.0001 compared to Pam (B), apoSAA (C), or control (D). ### p < 0.001 compared to unfiltered Pam+hSAA1 (D).

Article Snippet: Human SAA1 from Origene produced in HEK cells (hSAA1) or ovalbumin (Ova) was unexposed or exposed to Pam3CSK4 (Pam) overnight (control), and some of the preparation was subjected to concentration of proteins >3 kDa using concentrated using Amicon Ultra centrifugal concentrators (filtered).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Produced, Concentration Assay