Journal: PLoS ONE

Article Title: Pretransplant Infusion of Donor B Cells Enhances Donor-Specific Skin Allograft Survival

doi: 10.1371/journal.pone.0077761

Figure Lengend Snippet: ( A–B ) Cells from the LNs and spleen of naïve GFP + CBy mice were stained with mAbs against CD4, CD8, CD11c, Mac1, Lin (lineage markers, including CD3, CD19 and DX5) and NK1.1. Percent ( A ) and total number ( B ) of different subsets of donor cells were assessed by flow cytometry. ( C ) 2C F1 (anti-L d -2C-TCR + , H-2 b/d , L d− ) recipient mice were i.v. injected with 3×10 7 spleen and LN cells (▪, ▾) from GFP + CBy (H-2 b/d , L d+ ) donors. Seven days later, recipient mice were transplanted with sex-matched CBy (L d+ , ▪, n = 15) or SJL (H-2 s+ , ▾, n = 15) skin grafts. 2C F1 mice that were transplanted with CBy skin grafts without pretransplant DLI were used as control (▴, no DLI, n = 15). Graft survival was monitored by visual inspection daily for the first 2 weeks and twice a week thereafter. Representative data are shown from at least three independent experiments.

Article Snippet: To enrich the DN Tregs, CD4 + and CD8 + T cells were depleted by incubating the cells with anti-CD4 (RL-172-4, rat IgM) and anti-CD8 (3.168, rat IgM) depleting mAbs on ice for 30 minutes, followed by incubation with Low-Tox Rabbit Complement (Cedarlane Laboratories Limited, Hornby, ON) at 37°C for 45 minutes.

Techniques: Staining, Flow Cytometry, Injection

Journal: PLoS ONE

Article Title: Pretransplant Infusion of Donor B Cells Enhances Donor-Specific Skin Allograft Survival

doi: 10.1371/journal.pone.0077761

Figure Lengend Snippet: 2C F1 mice were left untreated (no DLI, ○), or i.v. infused with either 3×10 7 CD19 + (▪,×) or CD19 − (▴, •) cells, or untouched naïve B cells (CD43 - CD4 - Ter119 − cells, Δ, □) from CBy mice. Seven days later, recipients were transplanted with either sex-matched L d+ CBy (▪, n = 9; ▴, n = 9; □, n = 10; ○, n = 10), or SJL skin allografts (×, n = 10; •, n = 10; Δ, n = 10). Graft survival was monitored for more than 100 days. The mean survival time was calculated for each treatment and the log-rank test was used to analyze significance.

Article Snippet: To enrich the DN Tregs, CD4 + and CD8 + T cells were depleted by incubating the cells with anti-CD4 (RL-172-4, rat IgM) and anti-CD8 (3.168, rat IgM) depleting mAbs on ice for 30 minutes, followed by incubation with Low-Tox Rabbit Complement (Cedarlane Laboratories Limited, Hornby, ON) at 37°C for 45 minutes.

Techniques:

Journal: PLoS ONE

Article Title: Pretransplant Infusion of Donor B Cells Enhances Donor-Specific Skin Allograft Survival

doi: 10.1371/journal.pone.0077761

Figure Lengend Snippet: (A–B) 2C F1 mice were infused with 3×10 7 purified B or non-B cells from CBy mice as described in . Cells were harvested from LNs and spleens of the recipients 7 days later, stained with mAbs against CD4, CD8, NK1.1 and 1B2 in combination with activation markers CD62L or Ly6A. (A) Representative histograms from one of three independent experiments show percentages of CD62L + and Ly6A + populations on CD4 - CD8 - NK1.1 - 1B2 + antigen-specific DN Tregs. (B) Bar graphs summarized the percentages of CD62L + and Ly6A + cells from 3 independent experiments. (C) 2C F1 DN Tregs were co-cultured with increasing doses of irradiated CBy (L d+ ) B cells that had previously been activated with 10 µg/ml LPS for 24 hours in the presence of either 2C F1 -TCR non-specific P1A (dashed line) or specific QL9 (solid line) peptides that bind to MHC class I – L d . Proliferation of DN Tregs was assessed by 3 H-TdR labelling for 18hrs after 3-days of coculture. (D) LPS activated B cells (solid line) and mDCs (dashed line) were stained with mAb specific for L d . Isotype control was used as a negative control (shaded histogram). The numbers show the MFI of L d expression on B cells (top) and mDCs (bottom). (E–F) 2C F1 DN Tregs were co-incubated with L d+ B cells (CD19 + ) or mDCs (CD11c + , MHCII high , CD86 high ). (E) Representative histograms show L d expression on the surface of DN Tregs after 48 hrs of co-incubation with either B cells (left panel) or mDCs (right panel). Isotype control was used as a negative control (shaded histogram). The numbers are MFI of the L d alloantigen expressed on the DN Tregs. (F) MFI of L d on the surface of 1B2 + DN Tregs at 1, 24, and 48 hours after coculture with each cell type. These experiments were repeated at least 3 times and the results were analyzed by Two-way ANOVA. 0.01<* p <0.05, *** p <0.0001

Article Snippet: To enrich the DN Tregs, CD4 + and CD8 + T cells were depleted by incubating the cells with anti-CD4 (RL-172-4, rat IgM) and anti-CD8 (3.168, rat IgM) depleting mAbs on ice for 30 minutes, followed by incubation with Low-Tox Rabbit Complement (Cedarlane Laboratories Limited, Hornby, ON) at 37°C for 45 minutes.

Techniques: Purification, Staining, Activation Assay, Cell Culture, Irradiation, Negative Control, Expressing, Incubation

Journal: PLoS ONE

Article Title: Pretransplant Infusion of Donor B Cells Enhances Donor-Specific Skin Allograft Survival

doi: 10.1371/journal.pone.0077761

Figure Lengend Snippet: ( A, B ) 2C F1 mice were i.v. injected with 3×10 7 B or non-B cells isolated from CBy mice. Cells were harvested from spleen and LNs on day 7, stained with anti-CD4, anti-CD8, anti-NK1.1 and anti-1B2 mAbs, then examined by flow cytometry. ( A ) Donor-specific DN Tregs and CD8 + T cells were gated on 1B2 + population. ( B ) Based on the flow cytometry data, 1B2 + DN Treg to1B2 + CD8 ratio from a total of 8 mice from 3 independent experiments was calculated. ( C ) DN Tregs were isolated from 2C F1 mice 7 days after infusion of either CBy B or non-B cells followed by restimulation ex vivo of either CBy or SJL B or non-B cells before being used as effectors. Activated anti-L d+ or anti-H-2 s CD8 + T cells were used as targets. Effectors and targets were cultured either alone or together at varying ratios as indicated. After 24 hrs, cultures were stained with anti-1B2, anti-CD8 mAbs, Annexin V and PI. Percentages of Annexin V + and PI + cells in 1B2 + CD8 + gated cells are shown in ( C ). Data shown in ( D ) are from 3 independent experiments. Two-way ANOVA with Bonferroni's post-test was used to analyze the data. The asterisk symbol (*) shows significance between B (CBy) and Non-B (CBy) graphs. The hash symbol (#) shows significance between B (CBy) and B (SJL) graphs. *p<0.05, **p<0.01, ***p<0.001

Article Snippet: To enrich the DN Tregs, CD4 + and CD8 + T cells were depleted by incubating the cells with anti-CD4 (RL-172-4, rat IgM) and anti-CD8 (3.168, rat IgM) depleting mAbs on ice for 30 minutes, followed by incubation with Low-Tox Rabbit Complement (Cedarlane Laboratories Limited, Hornby, ON) at 37°C for 45 minutes.

Techniques: Injection, Isolation, Staining, Flow Cytometry, Ex Vivo, Cell Culture

Journal: Frontiers in Immunology

Article Title: Dim Light at Night Impairs Daily Variation of Circulating Immune Cells and Renal Immune Homeostasis

doi: 10.3389/fimmu.2020.614960

Figure Lengend Snippet: Dim light at night (dLAN) disturbs the daily variation and alters the number of circulating immune cells. (A–C) Flow cytometric analysis of peripheral white blood cells (WBCs) collected at ZT9 and ZT21 from rats exposed to either the control light–dark (LD) regime (CTRL) or dLAN (~2 lx) for 2 (upper rows) and 5 (lower rows) weeks. Data represent the mean ± SEM with n = 6–9 per group. (B) Numbers of CD4 + helper (Th) and CD8 + cytotoxic (Tc) T cells and the CD4/CD8 ratio. (C) Numbers of classical (CD43 lo HIS48 hi ) and non-classical (CD43 lo HIS48 hi ) monocytes. Significant differences were evaluated by two-way repeated ANOVA with the Tukey post hoc test for multiple comparisons. Only significant main effects (ZT and dLAN) or interactions (ZT×dLAN) are shown. Dotted lines indicate significant differences between individual groups if an interaction was significant. # P < 0.1, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The following fluorochrome-conjugated monoclonal antibodies were used: PE-Cy7 anti-rat CD45 (clone OX-1; Sony Biotechnology, USA; 1611065), FITC anti-rat CD3 (clone G4.18; Thermo Fisher Scientific, USA; 11-0030), APC anti-rat CD4 (clone OX-35; Thermo Fisher Scientific; 17-0040), PE anti-rat CD8a (clone OX-8; Thermo Fisher Scientific; 12-0084), PE anti-rat CD45RA (clone OX-33; Thermo Fisher Scientific; MR6404), APC anti-rat CD161a (clone 10/78; Thermo Fisher Scientific; MR6805), FITC anti-rat granulocytes (clone HIS48; Thermo Fisher Scientific; 11-0570), and PE anti-rat CD43 (clone W3/13; Sony Biotechnology; 1614060).

Techniques: Control

Journal: Journal of Neuroinflammation

Article Title: Perturbing chondroitin sulfate proteoglycan signaling through LAR and PTPσ receptors promotes a beneficial inflammatory response following spinal cord injury

doi: 10.1186/s12974-018-1128-2

Figure Lengend Snippet: List of antibodies used in this study

Article Snippet: CD4 , Cedarlane (Sheep, AF6439) , WB , 1:100.

Techniques: Blocking Assay

Journal: Journal of Neuroinflammation

Article Title: Perturbing chondroitin sulfate proteoglycan signaling through LAR and PTPσ receptors promotes a beneficial inflammatory response following spinal cord injury

doi: 10.1186/s12974-018-1128-2

Figure Lengend Snippet: List of flow cytometry antibodies used in this study

Article Snippet: CD4 , Cedarlane (Sheep, AF6439) , WB , 1:100.

Techniques: Flow Cytometry

Journal: Journal of Neuroinflammation

Article Title: Perturbing chondroitin sulfate proteoglycan signaling through LAR and PTPσ receptors promotes a beneficial inflammatory response following spinal cord injury

doi: 10.1186/s12974-018-1128-2

Figure Lengend Snippet: ILP and ISP promotes a phenotypic switch in helper T cells toward a T reg phenotype. a , b Flow cytometric assessment revealed no apparent difference in the overall infiltration of helper T cells (CD45 + /CD3 + /CD4 + ) in the injured spinal cord at 3 and 7 days post-injury between vehicle and ILP/ISP-treated animals. c , d However, ILP/ISP-treated animals exhibited a significant decrease in the number of effector T cells (CD45 + /CD3 + /CD4 + /IFNγ + ) at 7 days post-injury. e , f A significant increase in the total number of regulatory T cells (CD45 + /CD3 + /CD4 + /IL10 + ) was observed at 3 days post-injury in ILP/ISP-treated animals. g , h Representative flow cytometry gates are shown. i Western blot analysis showed upregulation of CD4 protein expression, at 7 and 14 days post-SCI compared to uninjured control group confirming infiltration of helper T cells in the injured spinal cord. Confirming our flow cytometry, ILP and ISP had no apparent effect on the overall protein expression of CD4. j However, ILP and ISP significantly increased FOXP3 protein expression, a marker of regulatory T cells, at both 7 and 14 days post-SCI compared to SCI vehicle control. Western blot results have been normalized to the actin loading control prior to subsequent normalization to the control values. The data show mean ± SEM, * p < 0.05, Student t test ( a – f ), one-way ANOVA ( i , j ), N = 4-6/group

Article Snippet: CD4 , Cedarlane (Sheep, AF6439) , WB , 1:100.

Techniques: Flow Cytometry, Western Blot, Expressing, Marker

Journal: Physiological Genomics

Article Title: Mycophenolate mofetil prevents cerebrovascular injury in stroke-prone spontaneously hypertensive rats

doi: 10.1152/physiolgenomics.00110.2016

Figure Lengend Snippet: Renal immune cell infiltration. Immunohistochemistry was used to detect CD43+ lymphocytes (top) and CD68+ macrophages (bottom) infiltration in the kidneys collected from vehicle-treated (−MMF) or MMF-treated (+MMF) rats at the end of the treatment protocol or at the time of euthanasia. Representative images are shown above. Scale bar = 50 μm for CD43 and 20 μm for CD68 images. A large number of infiltrating CD43+ and CD68+ cells (indicated by arrows) were detected in the kidneys of vehicle-treated SHR-A3 rats. Immune cell infiltration was markedly reduced by immunosuppression with MMF for 8 wk; n = 8–9. *P < 0.05 vs. vehicle-treated (−MMF) rats.

Article Snippet: CD68 (clone ED1, ABD Serotec) was used as a marker of activated microglia/macrophages, and CD43 (clone W3/13, ThermoFisher Scientific) was used to identify lymphocytes.

Techniques: Immunohistochemistry