Journal: Innate immunity

Article Title: Identification of CD300a as a new hypoxia-inducible gene and a regulator of CCL20 and VEGF production by human monocytes and macrophages.

doi: 10.1177/1753425913507095

Figure Lengend Snippet: Figure 6. Differential modulation of CCL20 and VEGF production by CD300a cross-linking on primary Mn. Mn were seeded onto plates precoated with a specific anti-CD300a agonist mAb (+) or a control IgG1 (–) and goat anti-mouse IgG-F(ab0)2, and cultured for 24 h under hypoxic conditions (Hypo, +) or in the presence of DFX (+). Cells were harvested and analyzed for (A) CCL20 and (C) VEGF mRNA expression by qRT-PCR. Relative transcript expression was calculated as detailed in the legend to Figure 1B. Data shown are expressed as fold increase relative to control Ig-cross-linked cells (considered equal to 1) and are representative of one of three experiments performed. (B) CCL20 and (D) VEGF content in conditioned medium were assayed by ELISA. Results are expressed as pg/106 cells/ml and represent the mean SEM of three different experiments. Values significantly different from those of cells cross- linked with isotype-matched Ab according to the Student’s t-test: *P 0.05; **P 0.01.

Article Snippet: Flow cytometry was performed as described.38 For the detection of surface markers, cells were re-suspended with FACS buffer (PBS supplemented with 0.2% BSA, 0.01% NaN3) and incubated with the fluorochrome-conjugated mAbs, anti-CD14-PE and antiCD300a (IRp60, clone P192 produced as described in Cantoni et al.14), the isotype-matched control Abs, mouse IgG2a-PE (Biolegend, Campoverde, Milano, Italy) and mouse IgG2a (R&D Systems, Space Import Export, Milano, Italy), and the secondary FITC-conjugated goat anti-mouse Ab (Invitrogen Life Technologies, Monza, Italy) for 30min at 4 C, after blocking nonspecific sites with rabbit IgG (Sigma, Milano, Italy).

Techniques: Control, Cell Culture, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Innate immunity

Article Title: Identification of CD300a as a new hypoxia-inducible gene and a regulator of CCL20 and VEGF production by human monocytes and macrophages.

doi: 10.1177/1753425913507095

Figure Lengend Snippet: Figure 7. CCL20 and VEGF production regulation by CD300a triggering on MDMs. MDMs were incubated with anti-CD300a Ab (+) or control isotype IgG1 (–) and goat anti-mouse IgG-F(ab0)2 for 24 h under hypoxic conditions (Hypo, +) or in the presence of DFX (+). (A) CCL20 mRNA expression, (C) VEGF mRNA expression and (B, D) cytokine release into the supernatant were analyzed 24 h after stimulation, as detailed in the legend to Figure 6. Values significantly different from those of cells cross-linked with isotype- matched Ab according to the Student’s t-test: * P 0.05.

Article Snippet: Flow cytometry was performed as described.38 For the detection of surface markers, cells were re-suspended with FACS buffer (PBS supplemented with 0.2% BSA, 0.01% NaN3) and incubated with the fluorochrome-conjugated mAbs, anti-CD14-PE and antiCD300a (IRp60, clone P192 produced as described in Cantoni et al.14), the isotype-matched control Abs, mouse IgG2a-PE (Biolegend, Campoverde, Milano, Italy) and mouse IgG2a (R&D Systems, Space Import Export, Milano, Italy), and the secondary FITC-conjugated goat anti-mouse Ab (Invitrogen Life Technologies, Monza, Italy) for 30min at 4 C, after blocking nonspecific sites with rabbit IgG (Sigma, Milano, Italy).

Techniques: Incubation, Control, Expressing

Journal: Tissue Engineering. Part A

Article Title: Human Endometrial Mesenchymal Stem Cells Modulate the Tissue Response and Mechanical Behavior of Polyamide Mesh Implants for Pelvic Organ Prolapse Repair

doi: 10.1089/ten.tea.2013.0170

Figure Lengend Snippet: Antibodies Used for Flow Cytometry

Article Snippet: The percentage of positive cells was based on IgG isotype antibodies used for control setting of gates. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Primary antibody Clone Concentration (μg/mL) Isotype Supplier CD29 mAb13 10 Rat IgG2a BD Pharmingen CD31 M89D3 10 Mouse IgG2a BD Pharmingen CD44 G44–26 10 Mouse IgG2b BD Pharmingen CD45 HI30 10 Mouse IgG1 CALTAG Laboratories CD73 AD2 10 Mouse IgG1 BD Pharmingen CD90 5E10 10 Mouse IgG1 BD Pharmingen CD105 266 10 Mouse IgG1 BD Pharmingen CD140b (PDGFRβ) PR7212 25 Mouse IgG1 R&D Systems CD146 CC9 25 Mouse IgG2a Donated by P. Simmons, Peter MacCallum Cancer Centre W5C5 W5C5 25 Mouse IgG1 Donated by Dr. Hans-Jörg Bühring, Tübingen University Open in a separate window Antibodies Used for Flow Cytometry Cell labeling Before implantation, P6 cells were labeled with VybrantTM DiO reagent (Life Technologies) according to the manufacturer's instructions.

Techniques: Concentration Assay

Journal: Tissue Engineering. Part A

Article Title: Human Endometrial Mesenchymal Stem Cells Modulate the Tissue Response and Mechanical Behavior of Polyamide Mesh Implants for Pelvic Organ Prolapse Repair

doi: 10.1089/ten.tea.2013.0170

Figure Lengend Snippet: Details of Antibodies Used for Immunohistochemistry

Article Snippet: The percentage of positive cells was based on IgG isotype antibodies used for control setting of gates. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Primary antibody Clone Concentration (μg/mL) Isotype Supplier CD29 mAb13 10 Rat IgG2a BD Pharmingen CD31 M89D3 10 Mouse IgG2a BD Pharmingen CD44 G44–26 10 Mouse IgG2b BD Pharmingen CD45 HI30 10 Mouse IgG1 CALTAG Laboratories CD73 AD2 10 Mouse IgG1 BD Pharmingen CD90 5E10 10 Mouse IgG1 BD Pharmingen CD105 266 10 Mouse IgG1 BD Pharmingen CD140b (PDGFRβ) PR7212 25 Mouse IgG1 R&D Systems CD146 CC9 25 Mouse IgG2a Donated by P. Simmons, Peter MacCallum Cancer Centre W5C5 W5C5 25 Mouse IgG1 Donated by Dr. Hans-Jörg Bühring, Tübingen University Open in a separate window Antibodies Used for Flow Cytometry Cell labeling Before implantation, P6 cells were labeled with VybrantTM DiO reagent (Life Technologies) according to the manufacturer's instructions.

Techniques: Concentration Assay, Labeling, Polymer, Marker

Journal: bioRxiv

Article Title: Microglial signalling pathway deficits associated with the R47H TREM2 variant linked to AD indicate inability to activate inflammasome

doi: 10.1101/2020.09.08.288043

Figure Lengend Snippet: a. Western blot analysis of signalling pathways in iPS-derived myeloid progenitor cells following TREM2 activation with TREM2 antibody crosslinking, or IgG control, in control and TREM2 R47H het patient lines. b. Quantification of pSYK, pERK1/2 and pAKT protein normalised to beta-actin; data are mean ± SEM, n=3-6 cell lines from 3 individual experiments, * P <0.05 (two-way ANOVA with Tukey’s correction), ns, non-significant.

Article Snippet: Myeloid progenitor cells were treated in suspension for 1 h with 5μg of IgG2B isotype control (clone 141945; R&D Systems) or αTREM2 (rat clone 237920; R&D Systems) before being treated with 10μg F(ab’)2 (Jackson #712006153) for 2 min of crosslinking.

Techniques: Western Blot, Derivative Assay, Activation Assay, Control