Journal: Frontiers in Immunology

Article Title: Extracellular CIRP-Impaired Rab26 Restrains EPOR-Mediated Macrophage Polarization in Acute Lung Injury

doi: 10.3389/fimmu.2021.768435

Figure Lengend Snippet: Rab26 deficiency reduces EPOR signaling and restrains macrophage polarization. A qPCR assay was conducted to evaluate the mRNA expression of EPOR (A) and PPARγ (B) in WT BMDMs treated with rhEPO (20 IU/ml) for the indicated time (n = 3). (C) The MFIs of cell surface EPOR in BMDMs treated with rhEPO (20 IU/ml) for the indicated times were tested by FACS (n = 3). * P < 0.05, ** P < 0.01 vs. 0 h. (D) The MFIs of CD80 and CD86 were evaluated by FACS in WT and Rab26 -/- BMDMs treated with or without eCIRP (1 µg/ml) or rhEPO (20 IU/ml) for 24 h (n = 3). (E) A qPCR assay was conducted to evaluate the mRNA expression of TNF-α, IL-6, and IL-1β in WT and Rab26 -/- BMDMs treated with eCIRP (1 µg/ml) and rhEPO (20 IU/ml) for 24 h (n = 3). (F) Localization of EPOR in WT and Rab26 -/- BMDMs. WT and Rab26 -/- BMDMs were stained with an anti-EPOR antibody (1:100 dilution) and Alexa Fluor 488-labeled secondary antibody (1:200 dilution) (green), and nuclei were stained with DAPI (blue). Representative confocal images of the surface and intracellular expression of EPOR are shown. Scale bar: 10 µm. Data are representative of at least two independent experiments. Results were expressed as mean ± SD. n. s., not statistically significant. * P < 0.05, ** P < 0.01. Statistics: One-way ANOVA with Tukey’s post-hoc test for multiple comparisons (A–E) . EPOR, erythropoietin receptor; PCR, polymerase chain reaction; PPAR, peroxisome proliferator-activated receptor; BMDM, bone marrow derived macrophage; EPO, erythropoietin; MFI, mean fluorescence intensity; FACS, fluorescence activated cell sorter; eCIRP, extracellular cold-inducible RNA-binding protein.

Article Snippet: Reagents were as follows: LPS from Escherichia coli O111:B4 (Sigma-Aldrich, #L4391), LPS from Escherichia coli 055:B5 (Sigma-Aldrich, #L2880), human CIRBP/CIRP (Sino Biological, #14578-H07E), rhEPO (Sunshine Pharmaceutical, Shenyang, China), cell dissociation buffer (Gibco, #13150016), PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, #23225), TRIzol Reagent (Sigma-Aldrich, #T9424), cOmpleteTM EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich, #04693159001), GoScriptTM Reverse Transcription System (Promega, #A2800), GoTaq ® qPCR Master Mix (Promega, #A6001), M-PER Protein Extraction Reagent (Thermo Fisher Scientific, #78510), PageRuler Prestained Protein Ladder (Thermo Fisher Scientific, #26616), Immobilon Western Chemiluminescent HRP Substrate (Millipore, #WBKLS0500), LEGENDplexTM Multi-Analyte Flow Assay Kit (BioLegend, #740740), Immunofluorescence Application Solutions Kit (CST, #12727), Anti-fade Reagent with DAPI (Coolaber, #SL 1841), and PE Annexin V Apoptosis Detection Kit (BD, #559763).

Techniques: Expressing, Staining, Labeling, Polymerase Chain Reaction, Derivative Assay, Fluorescence, RNA Binding Assay

Journal: Diagnostic Pathology

Article Title: PD1 hi cells associate with clusters of proliferating B-cells in marginal zone lymphoma

doi: 10.1186/s13000-018-0750-8

Figure Lengend Snippet: Workflow for immunohistochemistry and image analysis. a Diagram of the immunohistochemical workflow. b Quantification of the efficiency of elution for three antibodies: anti-PD1, anti-FOXP3 and anti-CD4. Black bars represent antibody staining intensity (mean ± SEM) and gray bars represent staining intensity following elution. Each experiment was carried out three times. The dotted line is the background intensity produced by an unstained tissue section. c Flow diagram to show Visiopharm methodology ( www.visiopharm.com ) used to analyse tissue sections stained sequentially with anti-CD4, anti-PD1 and anti-FOXP3 antibodies. The algorithm first identified cell nuclei by haematoxylin counterstain following which CD4 expressing cells were marked green. Next CD4 expressing cells were interrogated for co-expression of FOXP3. There was then a branch point in the algorithm. If FOXP3 expression was detected but there was no co-expression of PD1 at a high level (PD1 hi ), as previously defined by comparison with tonsillar Tfh cells, then the cell was determined to be Treg and the nucleus was labelled yellow. However, if the cell co-expressed CD4, FOXP3 and was PD1 hi then it was determined to be a Tfr cell and the nucleus was labelled turquoise. Cells that were CD4 expressing and PD1 hi , but not FOXP3 expressing were determined to be Tfh cells and the nuclei were labelled orange. Cells of each subset were then counted digitally. d Identification of specific T-cell subsets by multiplex immunohistochemistry. Tissue sections were stained with anti-CD4, anti-PD1 (EuroMabNet, clone NAT105) and anti-FOXP3 (EuroMabNet, clone 236A). Example Tfh cell (CD4 + PD1 + FOXP3 − ) painted orange, Treg cell (CD4 + PD1 − FOXP3 + ) painted yellow and Tfr cell (CD4 + PD1 + FOXP3 + ) painted turquoise are presented

Article Snippet: Visiopharm apps were developed (available on application to the authors) to paint Tfh cells (CD4 + PD1 hi FOXP3 − ) orange, Treg cells (CD4 + PD1 − FOXP3 + ) yellow and Tfr cells (CD4 + PD1 hi FOXP3 + ) turquoise (Fig. and ) prior to extracting cell numbers and location coordinates.

Techniques: Immunohistochemistry, Immunohistochemical staining, Staining, Produced, Expressing, Comparison, Multiplex Assay

Journal: Diagnostic Pathology

Article Title: PD1 hi cells associate with clusters of proliferating B-cells in marginal zone lymphoma

doi: 10.1186/s13000-018-0750-8

Figure Lengend Snippet: Architecture of CD4+ T-cell infiltration and T-cell gene signatures. a Tissue sections from 6 cases were stained with anti-CD20, anti-CD4 and anti-CD8. Surface plots generated in ImageJ ( https://imagej.net/Fiji ) are shown for each stain. Intensity has been normalised so that images can be compared. T-cell zones are evident and whilst these comprised both CD4 and CD8 T-cells, CD4 T-cells were found at higher density in 4 of 6 cases (#1, #2, #4 and #6). b Gene set enrichment analysis. Data from Arribas et al. (2012) 5 was employed to discover T-cell signatures. Utilising a false discovery rate (FDR < 0.25) for exploratory work. Two gene signatures were found to be positively enriched in MZL. NES is normalised enrichment score. c Support for use of PD1 hi and FOXP3 + as single surrogate markers of Tfh and Treg cells respectively. Numbers of Tfh and Treg cells, as defined (Fig. ), were compared against single marker expression of PD1 hi and FOXP3

Article Snippet: Visiopharm apps were developed (available on application to the authors) to paint Tfh cells (CD4 + PD1 hi FOXP3 − ) orange, Treg cells (CD4 + PD1 − FOXP3 + ) yellow and Tfr cells (CD4 + PD1 hi FOXP3 + ) turquoise (Fig. and ) prior to extracting cell numbers and location coordinates.

Techniques: Staining, Generated, Marker, Expressing

Journal: Diagnostic Pathology

Article Title: PD1 hi cells associate with clusters of proliferating B-cells in marginal zone lymphoma

doi: 10.1186/s13000-018-0750-8

Figure Lengend Snippet: Automated quantitation of marker proteins. a Automated quantitation of PD1 hi and FOXP3 + cells in 15 cases of MZL. Numbers above bars represent PD1 hi FOXP3 + ratio. b Analysis of cell density data and PD1 hi :FOXP3 + ratio by type of MZL: nodal, extranodal or splenic. Box and whisker plots show median, 90th and 10th centile. c Analysis of cell density data and PD1 hi :FOXP3 + ratio by stage of disease, either early stage (I and II) or advanced stage (III and IV)

Article Snippet: Visiopharm apps were developed (available on application to the authors) to paint Tfh cells (CD4 + PD1 hi FOXP3 − ) orange, Treg cells (CD4 + PD1 − FOXP3 + ) yellow and Tfr cells (CD4 + PD1 hi FOXP3 + ) turquoise (Fig. and ) prior to extracting cell numbers and location coordinates.

Techniques: Quantitation Assay, Marker, Whisker Assay

Journal: Diagnostic Pathology

Article Title: PD1 hi cells associate with clusters of proliferating B-cells in marginal zone lymphoma

doi: 10.1186/s13000-018-0750-8

Figure Lengend Snippet: Clustering of PD1 hi cells. a Left hand panels are density plots representing PD1 hi (top) and FOXP3 (bottom) for one case (lymph node tissue). The impression of greater clustering of PD1 hi cells is confirmed by statistical analysis using Ripleys K function (right hand panels). A measure of distance (r) on the x-axis is plotted against K(r) function on the y-axis. The curve for complete spatial randomness (CSR) (dotted red line) is compared to K function for MZL (solid black line) for PD1 (upper panel) and FOXP3 (lower panel). Greater upward deflection of the observed curve compared to CSR represents greater clustering with the difference between these curves calculated to provide an overall cluster score. b Cluster scores for MZL cases ( n = 15). PD1 hi cells are significantly more clustered than FOXP3 + cells (Mann-Whitney test, P < 0.01). c Immunofluorescence images. CD20 (green), Ki-67 (blue) and PD1 (red) staining of a lymph node from a patient with MZL. Low power (10×) views of the merged antibody stains show that proliferating cells occur in clusters. Three fields are shown as high power views (80×). Two of these fields are from regions that contain a high density of Ki-67 + cells (upper and lower rows) while the middle row is from a region of lower Ki-67 + density

Article Snippet: Visiopharm apps were developed (available on application to the authors) to paint Tfh cells (CD4 + PD1 hi FOXP3 − ) orange, Treg cells (CD4 + PD1 − FOXP3 + ) yellow and Tfr cells (CD4 + PD1 hi FOXP3 + ) turquoise (Fig. and ) prior to extracting cell numbers and location coordinates.

Techniques: MANN-WHITNEY, Immunofluorescence, Staining

Journal: Diagnostic Pathology

Article Title: PD1 hi cells associate with clusters of proliferating B-cells in marginal zone lymphoma

doi: 10.1186/s13000-018-0750-8

Figure Lengend Snippet: PD1 hi cells cluster with Ki-67 + cells. a Illustrative cartoon depicting low and high interactions with the respective Pearson correlation and Morisita-Horn index for both patterns. b Spatial interaction between FOXP3 or PD1 and Ki-67 cells. Scatter plots displaying Ki67 + against PD1 hi cells (upper panel) and FOXP3 + cells (lower panel) measured in spatial quadrants (200μm 2 ). Only quadrants containing at least 5 cells of any population are displayed. Quadrants containing higher densities of both cell types (> 50 cells) were considered to represent high interaction. 11.3% of all quadrants analysed for Ki67 + and PD1 hi cells met this definition of high interaction compared to only 1.6% of Ki67 + and FOXP3 + cells. Analysis was repeated for different sized quadrants (100 and 400μm 2 ) confirming similar results. c Nearest neighbour analysis. Distance from either FOXP3 + cells (upper panel) or PD1 hi cells (lower panel) was plotted for 10 5 cells sampled from 10 cases. For PD1 hi cells the large peak occurring at short distances to the nearest Ki-67 + cell indicates that these cells are closer to Ki-67 + cells than FOXP3 + cells, which are more evenly distributed. All clustering and spatial analysis was performed using the statistical programme R

Article Snippet: Visiopharm apps were developed (available on application to the authors) to paint Tfh cells (CD4 + PD1 hi FOXP3 − ) orange, Treg cells (CD4 + PD1 − FOXP3 + ) yellow and Tfr cells (CD4 + PD1 hi FOXP3 + ) turquoise (Fig. and ) prior to extracting cell numbers and location coordinates.

Techniques:

Journal: Stem Cell Reports

Article Title: Long-term adherence of human brain cells in vitro is enhanced by charged amine-based plasma polymer coatings

doi: 10.1016/j.stemcr.2022.01.013

Figure Lengend Snippet: Neuronal cells detach from standard glass surfaces sooner than from tissue culture polystyrene (A) Schematic of the cell culture microenvironment and modifications tested to improve neuronal attachment. (B) Human neural progenitor cells (NPCs) were pre-matured in BrainPhys + supplements for 0–2 weeks, replated on test surfaces, and further matured for up to 27 weeks in BrainPhys + supplements before phenotypic analyses. Before replating on test surfaces, post-mitotic precursors were sorted for DAPI exclusion at 2 weeks (see Experimental procedures). (C and D) Live neuronal cultures under phase contrast at 4× and 20× magnification, respectively. Detachment was quantified from 4× magnification phase contrast images by tracing regions with no cells (yellow) and calculating their combined area as a fraction of the total field of view area for each well. (E) Detachment on standard glass across multiple coating solutions: poly- l -ornithine (PLO; n = 4), PLO-laminin (Lam) (n = 6), Matrigel (n = 2), 24 h treatment of laminin (n = 6), 4 h treatment of laminin (n = 2), uncoated (n = 6), and poly- l -lysine (PLL; n = 2); n replicates from 2 independent experiments, means ± SEMs represented. (F) Cell detachment on standard glass compared to plastic (TCPS) both with PLO-Lam. Means ± SEMs from n = 5 biologically independent experiments, 2–6 replicates per experiment. (G and H) Spontaneous multi-electrode array (MEA) recordings of hPSC-derived neuronal cultures showing network burst activity and synchrony over 15–17 weeks on TCPS plates pre-coated with PLO-Lam. Means ± SEMs data from 16 replicate wells, each well containing 16 electrodes.

Article Snippet: For most of the experiments, NPCs were differentiated into post-mitotic neuronal cultures for 2 weeks in NMM, then live cell (DAPI) sorted (BD FACSMelody) and replated at 1.58 × 10 5 –2.1 × 10 5 cells/cm on TCPS, coated glass coverslips, or standard glass coverslips, in NMM containing half-concentrations of growth factors ( , , and , detachment; , morphology; and , , and , electrophysiology).

Techniques: Cell Culture, Derivative Assay, Activity Assay

Journal: Stem Cell Reports

Article Title: Long-term adherence of human brain cells in vitro is enhanced by charged amine-based plasma polymer coatings

doi: 10.1016/j.stemcr.2022.01.013

Figure Lengend Snippet: DAP-treated glass surface supports human proliferative neuronal/glia progenitors, brain tumor cells, and post-mitotic mature neurons and astrocytes (A–D) Analysis of proliferative human brain cells on various substrates. Attachment determined by counting fixed nuclei (DAPI + ) over time. Means ± SEMs fold change in number of nuclei per well (normalized to day 0) calculated for 5–6 replicate wells/condition/time point (1 independent experiment). All of the surfaces were pre-coated with Matrigel. Proliferative human brain cells analyzed were (A) patient-derived glioblastoma tumor cells (GBMs), (B) hESC-derived glial precursor cells (GPCs), and (C) hESC-derived neural progenitor cells (NPCs). (D) Immunofluorescence staining of DNA (DAPI; blue) and cell soma (CellTracker, red) at latest time point (8 days for brain cells). See also Figure S3 . (E) Proliferation of non-neuronal human embryonic stem cells (H9, hESC) was measured as the fraction of area covered by colonies normalized to day 0 (n = 5–6 replicates from 1 independent experiment). (F) Immunofluorescence staining of DNA (DAPI; blue) and cell soma (CellTracker, red) at latest time point (5 days for stem cells). See also Figure S3 . (G and H) Cytotoxicity analysis of neuronal cultures. All of the surfaces were pre-coated with PLO-Lam. (G) Lactate dehydrogenase (LDH) relative concentrations measured in neuronal culture supernatant (n = 15 replicates from 5 independent experiments) to estimate cellular stress. (H) Without fixation, DNA marker DAPI is incorporated in the nuclei of dead or dying cells (DAPI + ), but not live cells (DAPI − ). Means ± SEMs proportions of live cells (DAPI − ). (I) Immunofluorescence staining of MAP2, GFAP, and synapsin at 10× magnification (top) and 40× magnification (bottom) in neuronal cultures after 9 weeks in maturation medium, on glass-DAP-LAM. (J) Live neuronal cultures on glass-DAP-LAM under phase contrast at 20× after 27 weeks in maturation medium. (K) FACS analysis of the proportion of neurons (Lv-synapsin-GFP) and astrocytes (Lv-GFAP-tdTomato) at 9 weeks in maturation medium on different surfaces. For (H) and (K), n = 3–5 replicates per condition from 2 independent experiments. p values calculated using Mann-Whitney tests, ns for p > 0.05.

Article Snippet: For most of the experiments, NPCs were differentiated into post-mitotic neuronal cultures for 2 weeks in NMM, then live cell (DAPI) sorted (BD FACSMelody) and replated at 1.58 × 10 5 –2.1 × 10 5 cells/cm on TCPS, coated glass coverslips, or standard glass coverslips, in NMM containing half-concentrations of growth factors ( , , and , detachment; , morphology; and , , and , electrophysiology).

Techniques: Derivative Assay, Immunofluorescence, Staining, Marker, MANN-WHITNEY