Journal: Cell Reports Methods

Article Title: An efficient immunoassay for the B cell help function of SARS-CoV-2-specific memory CD4 + T cells

doi: 10.1016/j.crmeth.2022.100224

Figure Lengend Snippet: BAFF and SCGFβ are majorly implicated in the monocytes-driven enhanced B cell survival (A) Volcano plot shows the concentration difference versus −log10 adjusted p values for the comparison of secreted factors between B + M and B-alone conditions after 9 days. Statistical calculation was done by multiple t test corrected using the Bonferroni-Sidak method. Dotted line represents p = 0.05. Red open circles represent the significantly secreted analyte. (B) Dot plot shows the fold change of various secreted factors in the same conditions as mentioned in (A). Dotted line represents the fold change = 2. Red open circles represent the analyte with fold change of ≥2. Data are representative of n = 10 donors. (C) Flow cytometric plots show the live B cells (CD19 + ) in 6 days B + M cocultures in presence of isotype (mouse IgG) or various blocking conditions. (D) Bar plot shows the count of live B cells in various blocking conditions, mentioned in (C). (E) Percent survival of B cells (relative to B + M) in presence of isotype, αBAFF, or αBAFF + αSCGFβ blocking conditions. Data are shown as mean ± SEM. Data represent the pool of two independent experiments. Statistics are as follows: (D and E) one-way ANOVA followed by Bonferroni’s multiple comparisons test. See also Figure S8 .

Article Snippet: In some B cell survival assay, B cells were supplemented with either the recombinant BAFF (#2149-BF, R&D Systems) or SCGF (#1904-SC, R&D Systems) at various concentrations.

Techniques: Concentration Assay, Comparison, Blocking Assay

Journal: Cell Reports Methods

Article Title: An efficient immunoassay for the B cell help function of SARS-CoV-2-specific memory CD4 + T cells

doi: 10.1016/j.crmeth.2022.100224

Figure Lengend Snippet: Promoting B cell survival by growth factors BAFF and SCGF is not sufficient to replace monocyte supplementation in Ag-specific T-B cocultures (A) Flow cytometric dot plots show the live B cells (CD19 + ) in B alone (−) and B cells supplemented with SCGF (100 ng/mL), BAFF (10 ng/mL), BAFF + SCGF, and monocytes (M) after 6 days. B + M was used as the control for 100% survivability. (B) Count of live B cells in conditions mentioned in (A). (C) Line plot shows the percent survival of B cells (relative to B + M) in supplemented culture of B cells. (D) Flow cytometric contour plots show the analysis of plasma cells in T + B (−) and T + B supplemented with BAFF, BAFF + SCGF, and monocytes (M) in 9 days cocultures. (E) Plasma cell count in the conditions mentioned in (D). (F) Flow cytometric contour plots show the analysis of activated (ICOS + PD-1 + ) CD4 + T cells in the conditions mentioned in (D). (G) Count of activated T cells in the conditions mentioned in (D). (H and I) Dot plots show the background subtracted (H) plasma cell count and (I) activated T cell count in supplemented cocultures. (J) Flow cytometric contour plots show the AIM + (OX40 + CD25 + ) CD4 + T cells in T + B (−) or supplemented cocultures after 2 days. (K) Dot plots show the background subtracted AIM + CD4 + T cells. Data are represented as mean ± SEM, with each dot representing one donor. Data represent the pool of two independent experiments. Statistics are as follows: (B, C, H, I, and K) one-way ANOVA followed by Bonferroni’s multiple comparisons test and (E and G) two-tailed paired t test. See also Figures S9 and .

Article Snippet: In some B cell survival assay, B cells were supplemented with either the recombinant BAFF (#2149-BF, R&D Systems) or SCGF (#1904-SC, R&D Systems) at various concentrations.

Techniques: Control, Clinical Proteomics, Cell Counting, Two Tailed Test

Journal: Cell Reports Methods

Article Title: An efficient immunoassay for the B cell help function of SARS-CoV-2-specific memory CD4 + T cells

doi: 10.1016/j.crmeth.2022.100224

Figure Lengend Snippet:

Article Snippet: In some B cell survival assay, B cells were supplemented with either the recombinant BAFF (#2149-BF, R&D Systems) or SCGF (#1904-SC, R&D Systems) at various concentrations.

Techniques: Purification, Recombinant, Staining, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Software

Journal: eLife

Article Title: Integrin alpha11 is an Osteolectin receptor and is required for the maintenance of adult skeletal bone mass

doi: 10.7554/eLife.42274

Figure Lengend Snippet:

Article Snippet: Antibody , sheep polyclonal anti-human Osteolectin , R and D Systems , AF1904 , (1:1000).

Techniques: Recombinant, Diagnostic Assay, Cell Culture, Protease Inhibitor, Western Blot, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Fractionation

Journal: The EMBO Journal

Article Title: Mammalian adipogenesis regulator (Areg) cells use retinoic acid signalling to be non‐ and anti‐adipogenic in age‐dependent manner

doi: 10.15252/embj.2021108206

Figure Lengend Snippet: A Representative fluorescence microscopy images of CD142 − ASPCs co‐cultured with an empty transwell (T‐well) or with wild‐type (WT) CD142 + ASPCs after in vitro adipogenic differentiation. B Fraction of differentiated CD142 − ASPCs co‐cultured with an empty transwell (T‐well) or with wild‐type CD142 + ASPCs (non‐treated or treated with control scrambled siRNA) after in vitro adipogenic differentiation and as quantified by the “adiposcore”; marker shapes correspond to different biological replicates, n = 10, 4 biological replicates, 2–3 independent wells for each. C Mean size or number of quantified adipocytes in matrigel plugs which were implanted into the subcutaneous adipose depot after 3 weeks of high‐fat diet. The plugs were injected with either only CD142 − ASPCs, or with a mix containing 10–20% of CD142 + ASPCs. Markers correspond to different biological replicates, n = 12–36, 1–3 biological replicates (6 mice in total), 9–12 slides for each. D Representative histology images of matrigel implant cuts after fixation with 4% PFA and dehydration of plugs injected with CD142 − ASPCs (top) or 80% of CD142 − ASPCs and 20% of CD142 + ASPCs (bottom). Both images are of plugs dissected from a female mouse after matrigel injection and 3 weeks on high‐fat diet. In pink: matrigel, in white: adipocytes, scale bar = 500 μm, staining haematoxylin and eosin. E Expression heatmap listing genes linked to “negative regulation of fat cell differentiation” (GO:0045599) across bulk RNA‐seq samples of CD142 + and CD142 – ASPCs after adipogenic differentiation; the genes are ordered from top to bottom by the log 2 FC of CD142 + over CD142 − ASPCs after adipogenic differentiation; significantly differentially expressed genes (FDR < 0.05) are coloured in red; log normalised expression scaled by row; T‐well – transwell, scr – scrambled siRNA (control). F Representative fluorescence microscopy imaged of human‐derived stromal vascular fraction (SVF), containing the ASPCs, after in vitro adipogenic differentiation when co‐cultured with total, CD142 − or CD142 + mouse ASPCs. G Fraction of differentiated human‐derived SVF, containing the ASPCs co‐cultured with total, CD142 − or CD142 + mouse ASPCs after in vitro adipogenic differentiation shown in (E); marker shapes correspond to different biological replicates, n = 9, 3 biological replicates, 3 independent wells for each. H Bulk RNA‐seq‐derived expression plots of CD142 + ASPC (Areg) markers coding for secreted proteins that were selected for downstream validation: F3 (coding for CD142), Mgp (coding for Matrix Gla protein, MGP), Gdf10 (GDF10), Clec11a (CLEC11A), Cpe (Carboxypeptidase E, CPE) and Bgn (Biglycan, BGN), n = 4–7, 1–5 biological replicates, 1–4 independent wells for each. I Fraction of differentiated adult‐derived CD142 − ASPCs, as quantified by the “adiposcore”, treated with the indicated recombinant proteins shown in (J); marker shapes correspond to different biological replicates, n = 11–59, 2–10 biological replicates, 3–9 independent wells for each. J Representative fluorescence microscopy images of adult‐derived CD142 − ASPCs after in vitro adipogenic differentiation; the induction cocktail was supplemented with recombinant proteins corresponding to the selected Areg‐specific candidates: CD142, MGP, GDF10, CLEC11A, CPE and BGN at 100 ng/ml . Data information: In all images, nuclei are stained with Hoechst (blue) and lipids are stained with Bodipy (yellow); scale bars, 100 μm, bar colours: total ASPCs – brown, CD142 − ASPCs – yellow, CD142 + ASPCs (Aregs) – blue, recombinant BSA or DMSO treatment (negative controls) – light grey, recombinant EGF treatment (positive control) – dark grey. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, pairwise two‐sided t ‐test (B, C, G, I) or one‐way ANOVA and Tukey HSD post hoc test (H), for statistical details see .

Article Snippet: At confluence, the cells were treated with a single dose of classical white adipocyte differentiation induction cocktail at half of its usual concentration: 0.25 μM IBMX, 0.5 μM dexamethasone, 85 nM (0.5 μg/ml) insulin (in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin), supplemented with the following recombinant proteins: BSA (Sigma, # A3059, A9418), EGF (ThermoFisher Scientific, # PMG8043), CD142 (LifeSpan BioSciences, # LS‐G12283), MGP (LifeSpan BioSciences, #LS‐G13865), CLEC11A (R&D Systems, #3729‐SC), GDF10 (SinoBiological, #50165‐M01H), CPE (ArcoBioSystems, #CAE‐M5222) and BGN (R&D Systems, #8128‐CM) at various concentrations including all or some of: 10, 50, 100, 500, 750 and 1,000 ng/ml.

Techniques: Fluorescence, Microscopy, Cell Culture, In Vitro, Control, Marker, Injection, Staining, Expressing, Cell Differentiation, RNA Sequencing, Derivative Assay, Biomarker Discovery, Recombinant, Positive Control