Journal: Nature Communications

Article Title: Elucidating the fundamental fibrotic processes driving abdominal adhesion formation

doi: 10.1038/s41467-020-17883-1

Figure Lengend Snippet: a Parabiosis schematic. b H&E and IF data showing the presence of an abscess identified along the abdominal wall adjacent to the adhesion interface in a mouse parabiont, which is strongly GFP+ (green fluorescent protein) secondary to the presence of circulating immune cells in the abscess. c No GFP+ cells were identified in the adhesion interface. Green (GFP) represents circulating cells, purple (CD45) stains for immune cells. n = 3 biological replicates. Scale bars, 50 μm. d Schematic of the Actin CreER ::ROSA26 VT2/GK3 Rainbow mouse construct. e Schematic showing Actin CreER ::ROSA26 VT2/GK3 Rainbow mice locally induced with activated tamoxifen liposomes (LiTMX) at time of adhesion formation, using a published protocol , ERT2 estrogen receptor T2. f Actin CreER ::ROSA26 VT2/GK3 Rainbow mouse uninjured control (top panel) and adhesion tissue harvested at POD 14 (bottom panel). Clonal proliferation of fibroblasts are visualized along the adhesion interface. Representative samples, structures as labelled in figures, white dotted lines outline adhesion, white asterisk marks adhesion interface, confocal imaging. n = 5 biological replicates. Scale bars, 25 μm. g Schematic of the PDGFRA CreER ::ROSA26 VT2/GK3 Rainbow mouse construct. h Confocal imaging of representative PDGFRA CreER ::ROSA26 VT2/GK3 Rainbow mouse adhesion samples showing cellular clonality in the adhesion interface at postoperative day (POD) 7 (top panels, Imaris rendering at right), and POD 14 (bottom panels, Imaris rendering at right). Structures as labelled in figures, thick white dotted lines outline adhesion interface, thin white dotted lines outline individual clones, confocal imaging. n = 5 biological replicates. Scale bars, 50 μm.

Article Snippet: Antibodies used for FACS included: Anti-phospho-JUN (Cell Signaling, s73d47G9, lot: 5, used at 1:200), anti-phospho-STAT5-PECy7 (BD Biosciences, 560117, lot: 8266820, prediluted and used at manufacturer’s volume per test of 20 ul), anti-FSP-1 (Ray biotech, 188-11191, lot: 1804128, used at 10 ug/mL), IgG Pacific Blue (Thermo Fisher, p31582, lot: 1929717, used at 1 ug/mL), anti-PDGFRα (Abcam, ab90967, lot: gr321324-2, used at 10 ug/mL), IgG Alexa-Fluor 647 (Abcam, ab150159, lot: GR241187-2, used at 1:2000), IgG Alexa-Fluor 488 (Abcam, ab150077, lot: GR3224145-2, used at 1:2000), IgG Alexa-Fluor 647 (Abcam, ab: 150075, lot: GR269275-2, used at 1:2000), IgG Pacific Blue (Thermo Fisher, P10994, lot: 2045342, used at 1 ul/mL), anti-S100A4-PE (BioLegend, 370003; lot b286200, used at 1:20), anti-PDGFRa (Abcam, ab203491, lot: GR3226597-1, used at 1:50), anti-CD26 PECy7 (Biolegend, 302713, lot: B253866, used at 1:20), anti-CD45-FITC (Invitrogen, 11-9459-42, lot 4319940, used at 1:20), anti-Ter119-FITC (Invitrogen, 11-5921-85, lot :4322597, used at 2.5 ug/mL), anti-CD31-FITC (Thermo Fisher, 11-0311-82, lot: B224877, used at 1:100), anti-Tie2 (Thermo Fisher, 14-5987-82, lot: 2072830, used at 1:100), anti-CD324 (Biolegend, 147302, lot: B228369, used at 10 ug/mL), anti-CD326 (Biolegend, 118202, lot: B254013, used at 0.6 ug/mL), 488 secondary (Abcam, ab150157, used at 1:2000), anti-CD45-eFluor 450 (Invitrogen, 48-0451-82, lot: 1936503, used at 5 ug/mL), anti-Ter119-Pacific Blue (Invitrogen, 48-5921-82, lot: 1974934, used at 5 ug/mL), anti-CD31-eFluor 450 (Invitrogen, 48-0311-82, lot: 1982691, used at 2.5 ug/mL), anti-Tie2-biotin (Invitrogen, 13-5978-82, lot: 4304957, used at 5 ug/mL), anti-CD324-biotin (Invitrogen, 13-3249-82, lot:1916204, used at 2.5 ug/mL), eFluor 450-Streptavidin (Invitrogen, 48-4317-82, lot 1988686, used at 2.5 ug/mL), anti-CD326-eFluor 450 (Invitrogen, 48-5791-82, lot: 1984115, used at 10 ug/mL), anti-CD45-PECy7 (Thermo Fisher, MHCD4512, used at 1:100), anti-Ter119-PECy7 (Invitrogen, 25-5921-82, lot: 1994153, used at 5 ug/mL), anti-CD31-PECy7 (Invitrogen, 25-0311-81, lot 4318668, used at 5 ug/mL), anti-Tie2 (Invitrogen, 14-5987-82, lot: 2072830, used at 10 ug/mL), anti-CD326-PECy7, (BioLegend, 324221, lot: B266928, used at 1:20), and anti-CD324-PECy7 (Biolegend, 147310, lot: B255274, used at 2.4 ug/mL).

Techniques: Construct, Imaging, Clone Assay

Journal: Frontiers in Veterinary Science

Article Title: Characterisation of canine CD34+/CD45 diminished cells by colony-forming unit assay and transcriptome analysis

doi: 10.3389/fvets.2022.936623

Figure Lengend Snippet: Typical plot profiles of canine haematopoietic stem and progenitor cells (HSPCs). Canine bone marrow-derived mononuclear cells (cBM-MNCs) were stained with 7-aminoactinomycin D (7-AAD), which is a viability dye, phycoerythrin (PE)-labelled anti-canine CD34 monoclonal antibody (mAb), and fluorescein isothiocyanate (FITC)-labelled anti-canine CD45 mAb. (A) Whole viable cells, (B) CD34+ cells, and (C) CD34+/CD45 diminished (CD45dim) cells as analysed by flow cytometry.

Article Snippet: Phycoerythrin-conjugated mouse anti-canine CD34 (clone 1H6, BD Bioscience, diluted 1:20) and fluorescein-isothiocyanate-conjugated rat anti-canine CD45 (clone YKIX716.13, Bio-Rad, Hercules, CA, diluted 1:20) monoclonal antibodies were used for flow cytometry analysis.

Techniques: Derivative Assay, Staining, Flow Cytometry

Journal: Frontiers in Veterinary Science

Article Title: Characterisation of canine CD34+/CD45 diminished cells by colony-forming unit assay and transcriptome analysis

doi: 10.3389/fvets.2022.936623

Figure Lengend Snippet: Haematopoietic colony formation by canine haematopoietic stem and progenitor cells (HSPCs). (A) Microscopic observation of canine haematopoietic colonies (Scale bars: 200 μm). (B) Haematopoietic colonies for every 1,000 cells, as counted for whole viable cells, CD34+ cells, and CD34+/CD45diminished (CD45 dim ) cells. Experiments were performed four times independently. Results are presented as mean ± standard error of the mean (SEM). Statistical significance of total colony counts was assessed by one-way analysis of variance (ANOVA). * p < 0.05, *** p < 0.001.

Article Snippet: Phycoerythrin-conjugated mouse anti-canine CD34 (clone 1H6, BD Bioscience, diluted 1:20) and fluorescein-isothiocyanate-conjugated rat anti-canine CD45 (clone YKIX716.13, Bio-Rad, Hercules, CA, diluted 1:20) monoclonal antibodies were used for flow cytometry analysis.

Techniques:

Journal: Frontiers in Veterinary Science

Article Title: Characterisation of canine CD34+/CD45 diminished cells by colony-forming unit assay and transcriptome analysis

doi: 10.3389/fvets.2022.936623

Figure Lengend Snippet: Global gene expression profiling of canine haematopoietic stem and progenitor cells (HSPCs). (A) Principal component analysis of the global gene expression of canine HSPCs. Gene expression is demonstrated by dots and circles of red (whole viable cells), green (CD34+ cells), and blue [CD34+/CD45 diminished (CD45dim) cells]. (B) The volcano plot shows the results of differentially expressed gene (DEG) analysis between CD34+/CD45dim cells and CD34+ cells. Statistical significance was set as false discovery rate (FDR) <0.1. (C) Gene ontology (GO) and Kyoto Encyclopaedia Gene and Genomes (KEGG)-based pathway enrichment analysis of the downregulated genes of CD34+/CD45dim cells. The candidate terms of biological processes are illustrated with adjusted p -values < −Log10 16 (top). The candidate terms revealed by KEGG pathway analysis are demonstrated (bottom). (D) The volcano plot shows the results of DEG analysis between CD34+/CD45dim cells and whole viable cells. Statistical significance was set as FDR <0.1.

Article Snippet: Phycoerythrin-conjugated mouse anti-canine CD34 (clone 1H6, BD Bioscience, diluted 1:20) and fluorescein-isothiocyanate-conjugated rat anti-canine CD45 (clone YKIX716.13, Bio-Rad, Hercules, CA, diluted 1:20) monoclonal antibodies were used for flow cytometry analysis.

Techniques: Gene Expression

Journal: Journal of Neurotrauma

Article Title: Characterization of the Antibody Response after Cervical Spinal Cord Injury

doi: 10.1089/neu.2016.4498

Figure Lengend Snippet: Changes in splenic antibody-secreting cell (ASC) counts following cervical spinal cord injury (cSCI). (A) Representative brightfield microscopy images showing splenic immunoglobulin M (IgM)+ and IgG+ ASCs (or plasma cells) from age-matched naïve, sham, and SCI rats at 2 weeks post-injury. Red arrows indicate ASCs in the red pulp and blue arrows show ASCs in the white pulp of the spleen. Scale bar: 100 μm. (B) Total splenic ASC counts in naïve, sham, and SCI rats at 2, 10, and 20 weeks post-injury. At 2 weeks, the cSCI group had higher plasma cell counts than sham controls. Also, sham animals had higher counts of splenic ASCs than age-matched naïve rats. There was no significant difference in total plasma cell counts at subsequent time-points between animals with cSCI and sham injury or between naïve and sham animals, although ASC counts were higher in the cSCI group than in age-matched naïve controls at 10 weeks. *p < 0.05, one-way analysis of variance for each time-point with Bonferroni post hoc test, n = 6-8/group, mean ± standard error of the mean (SEM). (C) Changes in frequency of splenic B-cells at 2, 10, and 20 weeks post-injury. Top: Representative dot plot panels showing the gated population of B-lymphocytes out of viable CD45+ cells (leukocytes) in the spleen of a spinally injured and a sham rat at 2 weeks post-injury. Bottom: Changes in percent frequencies (%) of splenic B-cells at 2, 10, and 20 weeks post-injury. B-lymphocytes declined significantly at 2 weeks post-injury, compared with shams, but recovered to normal levels at subsequent time-points. *p < 0.05, Independent Student's t-test, n = 6/group, mean ± SEM. (D-E) Quantification of IgM+ and IgG+ ASCs in the spleen of naïve, sham and SCI rats at 2, 10, and 20 weeks post-injury. Asterisks indicate significant differences between SCI and sham groups only. (D) IgM+ ASC counts increased significantly at 2 and 10 weeks post-cSCI, compared with shams. At 20 weeks, there was no significant difference between groups. (E) IgG+ ASC counts increased at 2 weeks but declined significantly at 10 weeks post-cSCI, compared with shams. IgG+ ASC counts were similar between groups at 20 weeks. Color image is available online at www.liebertpub.com/neu

Article Snippet: Next, the samples were stained with fixable viability dye eFluor 780 (1:1000; Life Technologies) for 20 min at 4°C and subsequently with CD45-FITC (leukocytes; BD Biosciences), CD3-PE (T- lymphocytes; BD Biosciences) and CD45RA-V450 (B-lymphocytes; BD Biosciences).

Techniques: Microscopy

Journal: Journal of Neurotrauma

Article Title: Characterization of the Antibody Response after Cervical Spinal Cord Injury

doi: 10.1089/neu.2016.4498

Figure Lengend Snippet: Autoreactive T-dependent (TD) response in vitro following cervical spinal cord injury (cSCI). (A) Experimental workflow for the study of splenic T-cell proliferation in vitro and representative results indicating proliferation of T-cells when stimulated with injured spinal cord homogenate or Concanavalin A (ConA) at 2 weeks post-cSCI. Each peak of the histogram represents one cycle of cell division. The indicated percentage of cells in each cell cycle refers to the SCI group. (B) Proliferation (shown as division index) of splenic T-cells isolated from rats with cSCI or sham injury at 2, 10, and 20 weeks post-injury, after in vitro stimulation with homogenate from injured spinal cord at 2, 10, and 20 weeks post-injury, respectively. SCI-derived splenic T-cells proliferated more rapidly, compared with sham-derived splenocytes, at 2 weeks post-injury, but not at later time-points. (C) Proliferation of splenic T-cells isolated from rats with cSCI or sham injury at 2, 10, and 20 weeks post-injury after in vitro stimulation with ConA. At 2 weeks, SCI-derived T-cells showed less proliferation, compared with their sham counterparts. Contrary, at 10 weeks post-injury, SCI-derived splenic T-cells proliferated more than sham-derived T-cells. There was no significant difference at 20 weeks post-SCI. (D) Representative dot-plot image acquired with flow cytometry indicating the percent frequency (%) of splenic T-lymphocytes (CD3+) out of total viable leukocytes (CD45+) in SCI and sham rats at 2 weeks. (E) Changes in percent frequency (%) of splenic T-cells at 2, 10 and 20 weeks post-SCI. No significant alterations in T-cell frequency occur in the SCI group, compared with shams. Mann-Whitney test (B-C), Independent Student's t-test (E), *p < 0.05, n = 4/group (B-C), n = 6/group (D-E), mean ± standard error of the mean. (F) Development of germinal centers in the spleen of rats at 2 weeks post-cSCI. Representative image of a spleen section stained for Bcl-6 indicating a germinal center. The inset is a lower power image demonstrating the location of the germinal center within the white pulp of the spleen. Color image is available online at www.liebertpub.com/neu

Article Snippet: Next, the samples were stained with fixable viability dye eFluor 780 (1:1000; Life Technologies) for 20 min at 4°C and subsequently with CD45-FITC (leukocytes; BD Biosciences), CD3-PE (T- lymphocytes; BD Biosciences) and CD45RA-V450 (B-lymphocytes; BD Biosciences).

Techniques: In Vitro, Isolation, Derivative Assay, Flow Cytometry, MANN-WHITNEY, Staining

Journal: Aging Cell

Article Title: Compromised CD8+ T cell immunity in the aged brain increases severity of neurotropic coronavirus infection and postinfectious cognitive impairment

doi: 10.1111/acel.14409

Figure Lengend Snippet: Aging limits T RM establishment within the brain. 8 week. adult or 18 months aged C57BL/6 mice were inoculated with 10 3 pfu MHV‐A59 i.n., CD8 + T cells harvested from the spleen 7 DPI and then adoptively transferred intravenously (i.v.) via the tail vein into either 8 week. or 18 months old animals that were infected with (a,b) 10 4 pfu or (c,d) 10 3 pfu MHV‐A59 1 day prior. (a, c) Survival and (b, d) weight change were monitored for 30 DPI. (e) Representative flow cytometry plots of lymphocytes stained for CD8, then CD45.1 and CD45.2 to distinguish between host and donor cells isolated from the brains of 8 week. or 18 months old animals at 30 DPI following infection with 10 3 pfu MHV‐A59 and adoptive transfer of 8 week. or 18 months cells as indicated. (f) Frequency and (g) total number of total CD8 + cells and total transferred cells. (h) Representative flow cytometry histograms of CD103 expression by CD8 + T cells present in the brain of 8 week. or 18 months animals 30 DPI following infection and adoptive transfer as described. (i) Quantification of CD103 MFI by host or donor cells within the same host. Data are representative of 1 independent experiment with each data point representing an individual animal. Survival assessed by Log‐rank Mantel‐Cox assessment, weight change and flow cytometry assessment conducted according to two‐way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Tetramer staining was carried out at room temperature for 20 min in conjugation with other surface staining antibodies: CD8a (53–6.7, APC‐Cy7), CD4 (RM4‐5, APC), CD44 (IM7, PE‐Cy7), CD45 (30‐F11, APC), CD45.1 (20–0453, APC), CD45.2 (60–0454, PE‐Cy‐7), CD11b (M1/70, FITC), P2RY12 (S16007D, PE), CD68 (FA‐11, BV‐421), MHC II (M5/114.15.2, PerCP‐Cy5.5), obtained from Cytek Biosciences or eBioscience, then washed with 1× PBS and fixed with 2% paraformaldehyde (PFA).

Techniques: Infection, Flow Cytometry, Staining, Isolation, Adoptive Transfer Assay, Expressing

Journal: Aging Cell

Article Title: Compromised CD8+ T cell immunity in the aged brain increases severity of neurotropic coronavirus infection and postinfectious cognitive impairment

doi: 10.1111/acel.14409

Figure Lengend Snippet: CD8 + T cells mediate neuronal apoptosis following MHV‐A59 infection. (a) Representative IHC of DAPI, NeuN, and CD8a in the DG and cortex of 8 week or 18 months old animals at (a) 12 DPI and (b) 30 DPI. (c) Representative flow cytometry plots of CD45 + CD8 + cells stained for IFN‐γ isolated from the brains of 8 week or 18 months old, mock or MHV‐A59 infected animals at 30 DPI following 4 h. PMA/ionomycin stimulation. (d) Quantification of percent CD8 + CD44 + cells positive for IFN‐γ. (e) Representative immunocytochemical (ICC) staining for NeuN and TUNEL of primary cortical neurons following no treatment or infection with MHV‐A59 MOI 0.5, with or without coculture with CD8 + T cells purified from the spleen of an 8 week old animal at 7 DPI with 10 3 pfu MHV‐A59. Quantification of ICC images: (f) total number NeuN + neuronal nuclei, (g) total number TUNEL + NeuN + neuronal nuclei, (h) proportion of TUNEL + NeuN + neuronal nuclei of total NeuN + neuronal nuclei present. (i) Representative ICC of NeuN and TUNEL staining of primary embryonic cortical neurons following no treatment or infection with MHV‐A59 MOI 0.5, with or without naïve or PMA/ionomycin stimulated CD8 + T cells purified from the spleen of an 8 week old uninfected animal. Quantification of ICC images: (j) total number NeuN + neuronal nuclei, (k) total number TUNEL + NeuN + neuronal nuclei, and (l) proportion of TUNEL + NeuN + neuronal nuclei of total NeuN + neuronal nuclei present. Data are representative of three independent experiments with each data point representing an individual sample. All images were taken at 40X magnification and scale bar = 100 μm. Three images were captured per sample and averaged. NeuN + and TUNEL + NeuN + neuronal nuclei quantified in ImageJ software using the Cell Counter plugin. Statistics according to unpaired one‐way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Tetramer staining was carried out at room temperature for 20 min in conjugation with other surface staining antibodies: CD8a (53–6.7, APC‐Cy7), CD4 (RM4‐5, APC), CD44 (IM7, PE‐Cy7), CD45 (30‐F11, APC), CD45.1 (20–0453, APC), CD45.2 (60–0454, PE‐Cy‐7), CD11b (M1/70, FITC), P2RY12 (S16007D, PE), CD68 (FA‐11, BV‐421), MHC II (M5/114.15.2, PerCP‐Cy5.5), obtained from Cytek Biosciences or eBioscience, then washed with 1× PBS and fixed with 2% paraformaldehyde (PFA).

Techniques: Infection, Flow Cytometry, Staining, Isolation, TUNEL Assay, Purification, Software

Journal: PeerJ

Article Title: Effects of platelet-rich plasma on mesenchymal stem cells isolated from rat uterus

doi: 10.7717/peerj.10415

Figure Lengend Snippet: Flow cytometry analysis: (A) representative forward and side scattering dot plot with the region of interest (R1). The percentages of CD90, CD105, CD45, and CD34 positive cells in R1 are indicated (B). Green curve corresponds to the control (stained with secondary antibodies only). Anti-vimentin staining (C): negative control (secondary antibodies only) and immunocytochemistry with anti-vimentin antibodies (red) and the nuclei counterstained with DAPI (blue) are presented. Bars, 20 μm. Induced cell differentiation assay (D): control cells and cells following the induction of differentiation are presented. Adipogenic differentiation was revealed by Sudan III staining, osteogenic differentiation was revealed by alizarin red staining, and chondrogenic differentiation was revealed by alcian blue staining. Bars, 50 μm. One experimental block is marked by a dotted line.

Article Snippet: For immunostaining, 1 × 10 5 fixed cells were incubated in 100 μl of Rinsing Solution (Miltenyi Biotec, Somerville, MA, USA) with primary antibodies to CD90 (ab225, 1/100, Abcam), CD45 (130-107-846, clone REA504, 1/20, Miltenyi Biotec, Somerville, MA, USA), CD105 (ab107595, 1/100, Abcam), and CD34 (PAB18289, 1/100, Abnova) at RT for 1 h. Subsequently, the cells were incubated with secondary antibodies: anti-mouse Ig-FITC (ab6785, 1/500, Abcam) or anti-rabbit Ig-PE (sc-3739, 1/100, Santa Cruz) at RT for 1 h in the dark.

Techniques: Flow Cytometry, Control, Staining, Negative Control, Immunocytochemistry, Cell Differentiation, Blocking Assay