Journal: Bioactive materials

Article Title: Calcium phosphate-based materials regulate osteoclast-mediated osseointegration.

doi: 10.1016/j.bioactmat.2021.05.003

Figure Lengend Snippet: Fig. 5. Effect of Ca/P ratio in CPC on RANKL-RANK signaling. (a) Immunofluorescence analysis of RANK expression (red) in osteoclasts on surface of CPC scaffold. (b) Concentration of RANK was analysis by ELISA. The concentration of RANK was measured after inducing BMMs for 1 days. (c) Affinity between RANKL and RANK was measured in BIAcore T200 system. Human-RANK anchored on the chip was applied to interact with the human-RANKL dissolved into the CPC extract. The vehicle means MEM-α medium. (d) 1.67CPC promoted RANKL-induced phosphorylation of NF-κB p65, and degradation of IκBα. (e) p-p65 and (f) IκBα intensity of Western blot bands was calculated with normalizing to β-actin. The untreated medium was treated as control. (g–l) Relative mRNA expression of osteoclast was measured after inducing BMMs for 7 days in CPC extract. BMMs cultured with untreated medium was treated as control. (*P ＜ 0.05, **P ＜ 0.01,***P ＜ 0.001).

Article Snippet: The concentration of RANK was measured by Mouse RANK ELISA Kit (Boster Biological Technology, China), according to the manufacturer’s instructions and the total protein concentration was measured by BCA Protein Assay Kit (Beyotime, China), which was used to normalize the expression of RANK.

Techniques: Immunofluorescence, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Western Blot, Control, Cell Culture

Journal: Journal of Neuroinflammation

Article Title: Lentiviral vector-mediated stable expression of sTNFR-Fc in human macrophage and neuronal cells as a potential therapy for neuroAIDS

doi: 10.1186/1742-2094-8-48

Figure Lengend Snippet: Lentiviral vector mediated delivery and expression of sTNFR-Fc . (A) Schematic maps of the lentiviral transfer plasmids, pHR-hTNFR-Fc-eGFP and pHR-Fc-eGFP. LTR: long terminal repeat; ψ: packaging signal; SA, SD: splice donor, splice acceptor; CMV: cytomegalovirus promoter; hTNFR-Fc: codon-optimized gene encoding the extracellular domain of the human TNF receptor type 2, fused to the hinge domain from IgG1 and the Fc domain from human IgG3; Fc: hinge domain from IgG1 and the Fc domain from human IgG3; IRES: internal ribosome entry site; GFP: green fluorescent protein.

Article Snippet: The quantitation of sTNFR-Fc protein was based on the optical density values at 450 nm, compared with a standard curve of purified human sTNFR-Fc protein (R&D Systems, Recombinant Human TNF RII/TNFRSF1B/Fc Chimera), using an ELISA reader (Beckman Coulter AD340).

Techniques: Plasmid Preparation, Expressing

Journal: Journal of Neuroinflammation

Article Title: Lentiviral vector-mediated stable expression of sTNFR-Fc in human macrophage and neuronal cells as a potential therapy for neuroAIDS

doi: 10.1186/1742-2094-8-48

Figure Lengend Snippet: Stable and high level secretion of sTNFR-Fc in transduced cells . (A) Immunoblot detection of sTNFR-Fc protein in transfected cells. Mock = supernatant from mock transfected 293T cells; Intracellular = transfected cell lysate; and Secreted = supernatant from the transfected 293T cells. (B) Immunoblot detection of sTNFR-Fc protein in transduced cells intracellularly (cell lysate) and extracellularly (supernatant). HTB-T = transduced HTB-11 cells; CHME-T2 = CHME-5 cells transduced twice with the vector; CHME-T1 = CHME-5 cells transduced once with the vector. (C) Stable expression of sTNFR-Fc protein in conditioned medium of transduced cells. Expression of sTNFR-Fc in supernatants from transduced cells was measured by ELISA; results shown at different cell passages represent mean values from three independent experiments and error bars denote the standard deviation. (D) Stable expression of GFP in lentivirus vector-transduced cells. Transduced cells were passed in vitro and the percentage of GFP positive cells was determined every 5 passages by calculating the percentage of GFP+ cells within the culture using an inverted fluorescent microscope (Nikon Eclipse TE2000-U) with a digital camera attachment. Data presented here represent mean values from at least three independent experiments and error bars denote the standard deviation.

Article Snippet: The quantitation of sTNFR-Fc protein was based on the optical density values at 450 nm, compared with a standard curve of purified human sTNFR-Fc protein (R&D Systems, Recombinant Human TNF RII/TNFRSF1B/Fc Chimera), using an ELISA reader (Beckman Coulter AD340).

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation, In Vitro, Microscopy

Journal: Journal of Neuroinflammation

Article Title: Lentiviral vector-mediated stable expression of sTNFR-Fc in human macrophage and neuronal cells as a potential therapy for neuroAIDS

doi: 10.1186/1742-2094-8-48

Figure Lengend Snippet: Specific binding of expressed sTNFR-Fc to TNF-α by dot immunoblot assay . One microgram of standard recombinant human TNF-α protein was spotted onto nitrocellular membrane (NCM) and then blocked and incubated with supernatants from normal and transduced cells. After three washes with TBST, a goat-anti-human IgG Fc-HRP conjugate was applied and incubated at RT for 1 h. Specific binding was visualized by color deposition on the NCM following incubation with diaminobenzidine (DAB) substrate. Lanes: HTB-NT = supernatant from non-transduced HTB-11 cells and CHME-NT = supernatant from non-transduced CHME-5 cells used as negative controls; HTB-T = supernatant collected from transduced HTB-11 cells and CHME-T2 = supernatant from transduced CHME-5 cells; and C + = purified recombinant sTNFR-Fc protein as a positive control.

Article Snippet: The quantitation of sTNFR-Fc protein was based on the optical density values at 450 nm, compared with a standard curve of purified human sTNFR-Fc protein (R&D Systems, Recombinant Human TNF RII/TNFRSF1B/Fc Chimera), using an ELISA reader (Beckman Coulter AD340).

Techniques: Binding Assay, Western Blot, Recombinant, Membrane, Incubation, Purification, Positive Control

Journal: Journal of Neuroinflammation

Article Title: Lentiviral vector-mediated stable expression of sTNFR-Fc in human macrophage and neuronal cells as a potential therapy for neuroAIDS

doi: 10.1186/1742-2094-8-48

Figure Lengend Snippet: Functional antagonization of sTNFR-Fc against TNF-α . As described in materials and methods, TNF-α sensitive L929 cells were treated with TNF-α alone (80 ng/mL) or with TNF-α plus culture supernatants from vector-transduced cells; purified recombinant sTNFR-Fc protein (160 ng/mL) was used as a positive control. After incubation for 24 h, cell viability was then evaluated by MTT assay. Viability was significantly higher for the cells treated with conditioned medium from transduced cells expressing hTNFR-Fc (** p < 0.01 for HTB-T; *p < 0.05 for CHME-T) when compared to cultures that received TNF-α alone, or TNF-α plus culture supernatants from parental cells (CHME-N and HTB-N). Results shown represent mean levels of three independent experiments and error bars denote the standard deviation.

Article Snippet: The quantitation of sTNFR-Fc protein was based on the optical density values at 450 nm, compared with a standard curve of purified human sTNFR-Fc protein (R&D Systems, Recombinant Human TNF RII/TNFRSF1B/Fc Chimera), using an ELISA reader (Beckman Coulter AD340).

Techniques: Functional Assay, Plasmid Preparation, Purification, Recombinant, Positive Control, Incubation, MTT Assay, Expressing, Standard Deviation

Journal: Journal of Neuroinflammation

Article Title: Lentiviral vector-mediated stable expression of sTNFR-Fc in human macrophage and neuronal cells as a potential therapy for neuroAIDS

doi: 10.1186/1742-2094-8-48

Figure Lengend Snippet: sTNFR-Fc mediated protection of neuronal cells from TNF-α, HIV-Tat and gp120 . (A) Non-transduced human neuronal cells, HTB-11, were treated with TNF-α alone (80 ng/mL), or with TNF-α plus culture supernatants (1:10 dilution) from vector-transduced cells (as indicated). Cells were incubated at 37°C for 3 days, and viability of the cells was determined by the trypan blue exclusion assay. sTNFR-Fc = supernatant collected from HTB-11 or CHME-5 cells transduced with the hTNFR-Fc encoding vector; Fc = supernatant collected from HTB-11 or CHME-5 cells transduced with the vector encoding human Fc only; rTNFR = purified commercial recombinant TNFR-Fc protein (160 ng/mL), used as a positive control. Results shown represent mean levels of three independent experiments and error bars denote the standard deviation. (B) Vector transduced human HTB-11 cells were exposed to TNF-α as described in 7A. HTB-11 cells transduced either with the sTNFR-Fc or Fc encoding vector were used in this experiment, along with non-transduced cells as controls. Results shown represent mean levels of three independent experiments and error bars denote the standard deviation. (C) sTNFR-Fc protects primary rat neurons against HIV-1 Tat-mediated toxicity. Medium from vector-transduced or parental HTB-11 cells was collected at day 10, diluted and mixed with 500 nM HIV-1 Tat protein, prior to addition to primary rat neurons. Following 24 hours incubation at 37°C, these test cultures along with the control were analyzed by TUNEL assay. Cell death was significantly reduced in neuronal cultures that were treated with Tat plus conditioned medium from HTB-11 cells transduced with the sTNRF-Fc encoding lentiviral vector (HTB-T 10d) versus cells treated with Tat plus conditioned medium from parental HTB-11 cells (HTB 10d) (*p < 0.01). NT, normal neuronal cells received no Tat, and -, neuronal cells exposed to Tat as a positive control. Results shown represent mean levels of three independent experiments and error bars denote the standard deviation. (D) sTNFR-Fc mediated neuronal protection of human HTB-11 cells against HIV-1 gp120 toxicity. Conditioned media from hTNFR-Fc or Fc vector transduced HTB-11 cells were collected, diluted and mixed with 100 ng/mL (gp120A) or 250 ng/mL (gp120B) HIV-1 gp120 protein, with or without HIV-1 Tat, prior to addition to HTB-11 cells. Following 3 days incubation at 37°C, cell viability of these cultures, together with control cultures, was determined by trypan blue exclusion assay (***p < 0.001). Results shown represent mean levels of three independent experiments and error bars denote the standard deviation.

Article Snippet: The quantitation of sTNFR-Fc protein was based on the optical density values at 450 nm, compared with a standard curve of purified human sTNFR-Fc protein (R&D Systems, Recombinant Human TNF RII/TNFRSF1B/Fc Chimera), using an ELISA reader (Beckman Coulter AD340).

Techniques: Plasmid Preparation, Incubation, Trypan Blue Exclusion Assay, Transduction, Purification, Recombinant, Positive Control, Standard Deviation, TUNEL Assay

Journal: Nature immunology

Article Title: Blocking elevated p38 MAPK restores efferocytosis and inflammatory resolution in the elderly

doi: 10.1038/s41590-020-0646-0

Figure Lengend Snippet: List of antibodies used in flow cytometry.

Article Snippet: Stains were performed for 30 minutes at 4 o C and followed by two washes with PBS containing 2% FCS and 2 mM EDTA before fixation in 0.5% PFA in PBS. table ft1 table-wrap mode="anchored" t5 Table 2.2: caption a7 Name Supplier Type Clone CD3 Biolegend Mouse IgG2a HIT3a CD11b Biolegend Mouse IgG1 ICRF44 CD14 Biolegend Mouse IgG2a M5E2 CD14 Biolegend Mouse IgG1 HCD14 CD16 Biolegend Mouse IgG1 3G8 CD19 Biolegend Mouse IgG1 HIB19 CD36 BD Biosciences Mouse IgM CB38 (NL07) CD51 (ITGAV) Miltenyi Biotec Recombinant IgG1 REA181 CD56 Biolegend Mouse IgG2a MEM-188 CD62L Biolegend Mouse IgG1 DREG-56 CD66b Biolegend Mouse IgM G10F5 CD95 (Fas) Biolegend Mouse IgG1 DX2 CD120a (TNFRI) Miltenyi Biotec Recombinant IgG1 REA252 CD120b (TNFRII) Biolegend Rat IgG2a 3G7A02 CD163 Biolegend Mouse IgG1 RM3/1 CD181 (CXCR1) Biolegend Mouse IgG2b 8F1/CXCR1 CD182 (CXCR2) Biolegend Mouse IgG1 5E8/CXCR2 CD192 (CCR2) BD Biosciences Mouse IgG2a LS132.1D9 (1D9) CD197 (CCR7) Biolegend Mouse IgG2a G043H7 CD206 Biolegend Mouse IgG1 15-2 Siglec-8 Biolegend Mouse IgG1 837535 HLA-DR BD Biosciences Mouse IgG2a G46-6 MerTK eBioscience Rat IgG2a DS5MMER TIM-4 Biolegend Mouse IgG1 9F4 P-p38 eBioscience Mouse IgG2b 4NIT4KK p65 (RelA) Biolegend Mouse IgG2b 14G10A21 P-STAT-3 (Tyr705) Biolegend Mouse IgG1 13A3-1 Live/Dead Zombie NIR Biolegend N/A N/A Open in a separate window List of antibodies used in flow cytometry.

Techniques: Cytometry, Recombinant

Journal: Micromachines

Article Title: A Novel Cortisol Immunosensor Based on a Hafnium Oxide/Silicon Structure for Heart Failure Diagnosis

doi: 10.3390/mi13122235

Figure Lengend Snippet: ( A ) Example of Nyquist plots for the Randles equivalent circuit model obtained by analyzing cortisol standard solutions in PBS (2, 10, 15, and 50 ng mL −1 ). EIS frequency ranged from 5 Hz to 100 kHz, and a sinus amplitude of 25 mV with polarization potential of −1.5 V; ( B ) sensitivity curves obtained by analyzing standard solution containing cortisol or other HF biomarkers (e.g., TNF-α and NT-proBNP) in the concentration range 2–50 ng mL −1 using the HfO 2 substrate functionalized with anti-cortisol antibody.

Article Snippet: Recombinant human TNF-α (Cat. No. 210-TA) was supplied by BioTechne (R&DSystems, noyal chatillon sur seiche, France).

Techniques: Concentration Assay

Journal: Micromachines

Article Title: A Novel Cortisol Immunosensor Based on a Hafnium Oxide/Silicon Structure for Heart Failure Diagnosis

doi: 10.3390/mi13122235

Figure Lengend Snippet: ( A ) Capacitance–voltage plots for cortisol detection using the capacitance biosensor; ( B ) the calibration curves of the cortisol detection (black curve) and the two interferences: TNF-α (red curve) and NT-proBNP (blue curve).

Article Snippet: Recombinant human TNF-α (Cat. No. 210-TA) was supplied by BioTechne (R&DSystems, noyal chatillon sur seiche, France).

Techniques:

Journal: Micromachines

Article Title: A Novel Cortisol Immunosensor Based on a Hafnium Oxide/Silicon Structure for Heart Failure Diagnosis

doi: 10.3390/mi13122235

Figure Lengend Snippet: ( A ) Capacitance–voltage plots for TNF-α detection; ( B ) for NT-proBNP detection using the capacitive immunosensor.

Article Snippet: Recombinant human TNF-α (Cat. No. 210-TA) was supplied by BioTechne (R&DSystems, noyal chatillon sur seiche, France).

Techniques:

Journal: Immunity

Article Title: The Fc domain of immunoglobulin is sufficient to bridge NK cells with virally infected cells

doi: 10.1016/j.immuni.2017.06.019

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Detailed methods are provided in the online version of this paper and include the following: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies BV421 anti-human CD3 (clone HIT3a) BD Cat# 740073 APC-H7 anti-human CD14 (M5E2) BD Cat# 561384 PE anti-human CD19 (clone HIB19) BD Cat# 555413 PC7 anti-human CD56 (clone N901) Beckman Coulter Cat# {"type":"entrez-nucleotide","attrs":{"text":"A51078","term_id":"2303855","term_text":"A51078"}} A51078 FITC anti-human CD69 (clone FN50) BD Cat# 555530 PE anti-human CD62L (clone DREG-56) BD Cat# 555544 V421anti-human CD107a (clone H4a3) Biolegend Cat# 328626 APC/Cy7 anti-human CD16 (clone 3G8) Biolegend Cat# 302018 FITC CD64 (clone 10.1) BD Cat# 555527 PE Ms IgG1, κ (clone MOPC-21) BD Cat# 555749 APC anti-human IgG (polyclonal) Jackson ImmunoResearch Cat# 209605098 APC anti-human IFNgamma (clone B27) BD Cat# 554702 Anti-HSV1 gE (clone 9H3) abcam Cat# Ab6510 FITC anti-human CD3ζ (clone 6B10.2) Biolegend Cat# 644104 APC anti-human CD3zeta (pY142) (clone K25-407.69) BD Cat# 558489 Anti asialo gm1 Biolegend Cat# 146002 PE anti-human CD120a (TNF-RI) (clone REA252) Miltenyi Biotech Cat# 130-106-360 IgG1, Kappa from human myeloma plasma Sigma Cat# I5154-1MG IgG3 Kappa Sigma Cat# I5654 Immune globulin, GamaStan S/D GRIFOLS N/A Rituximab Genentech N/A Obinutuzumab Janssen N/A Immunoglobulin G-Fc Fragment; Purified Scripps Laboratories Cat# I2724 Bacterial and Virus Strains HSV1 F strain ATCC Cat# VR-733 HSV1 Us8- F strain Suenaga et al., 2014 N/A R8411 (HSV1 F strain expresses luciferase) Zerboni et al., 2013 N/A MCMV Orr MT et al., 2010 N/A Staphylococcus newman strain (wt) ATCC Cat# 25904 Staphylococcus newman strain (spa-) Patel et al., 1987 N/A Biological Samples Umbilical cord blood American Red Cross, Columbus, OH, USA N/A Chemicals, Peptides, and Recombinant Proteins FCGR3A (CD16a), 176 Phe/Val Recombinant Human Protein, His.

Techniques: Clinical Proteomics, Purification, Virus, Luciferase, Recombinant, Cell Isolation, Enzyme-linked Immunosorbent Assay, Gel Extraction, Magnetic Beads, Plasmid Preparation, Software