Journal: International Journal of Molecular Sciences

Article Title: Schwann Cell Transplantation Subdues the Pro-Inflammatory Innate Immune Cell Response after Spinal Cord Injury

doi: 10.3390/ijms19092550

Figure Lengend Snippet: SC transplantation shifted the CD11b immune cell population from an Arg1 − iNOS + pro-inflammatory to an intermediate Arg1 + iNOS + phenotype after SCI. Representative images of flow cytometry analysis and pie charts of CD11b population dynamics at 14 days post-injury (7 days post-transplantation) show, compared with SCI controls ( A – C ), a decreased percentage of CD11b cells stained with Arg1 − iNOS + and an increased percentage for Arg1 + iNOS + in animals receiving SC transplants ( D – F ). Results are expressed as mean ± standard deviation (SD). Abbreviations on the graphs are: Fluorescein isothiocyanate (FITC), Allophycocyanin (APC) and Forward Scatter (FSC-A). For panels ( B , E ), the blue dots represent the CD11b population that is iNOS − -Arg1 − , the orange dots represent the CD11b population that is iNOS + -Arg1 − and the green dots represent the CD11b population that is double positive for both iNOS + -Arg1 + . These colored dots are also shown in the forward scatter plots of panels ( A , D ).

Article Snippet: Cells were then washed once with flow cytometry staining buffer (R&D System), fixed, and permeabilized (BD Cytofix/Cytoperm, BD Bioscience) for 20 min to permit immunostaining with selected antibodies against intracellular proteins for 1 h on ice.

Techniques: Transplantation Assay, Flow Cytometry, Staining, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Schwann Cell Transplantation Subdues the Pro-Inflammatory Innate Immune Cell Response after Spinal Cord Injury

doi: 10.3390/ijms19092550

Figure Lengend Snippet: CD68 immune cells with a pro-inflammatory Arg1 − iNOS + phenotype after SCI are reduced by SC transplantation. Representative images of flow cytometry analysis and pie charts of CD68 population dynamics at 14 days post-injury (7 days post-transplantation) reveal that CD68 immune cells with a pro-inflammatory Arg1 − iNOS + phenotype after SCI ( A – C ) are reduced by the intraspinal transplantation of SCs ( D – F ). Results are expressed as mean ± SD. For panels ( A , D ), the orange dots represent the ED1 population that were gated based on their forward and side scatter from the total events (blue) that were acquired. The orange dots in the different quadrants of ( B , E ) represent the ED1 population that was positive for either Arg1 (top left quadrant) or iNOS (bottom right quadrant) or double positive for both markers (top right quadrant).

Article Snippet: Cells were then washed once with flow cytometry staining buffer (R&D System), fixed, and permeabilized (BD Cytofix/Cytoperm, BD Bioscience) for 20 min to permit immunostaining with selected antibodies against intracellular proteins for 1 h on ice.

Techniques: Transplantation Assay, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Schwann Cell Transplantation Subdues the Pro-Inflammatory Innate Immune Cell Response after Spinal Cord Injury

doi: 10.3390/ijms19092550

Figure Lengend Snippet: CD11b immune cells expressing a highly pro-inflammatory CD38 + iNOS + phenotype or anti-inflammatory Arg1 + CD163 + form after SCI were unaltered by SC transplantation. Representative images of flow cytometry analysis and pie charts of CD11b population dynamics at 14 days post-injury (7 days post-transplantation) reveal that CD11b immune cells with a highly pro-inflammatory CD38 + iNOS + phenotype after SCI ( A , B ) are not altered by SC transplantation ( E , F ). Similarly, CD11b immune cells with a highly anti-inflammatory Arg1 + CD163 + phenotype were unchanged across SCI controls ( C , D ) and SC-transplanted groups ( G , H ). Results are expressed as mean ± SD. For panels ( A , B ), the blue dots represent the CD11b population that were CD38 − iNOS − , the orange dots represent the CD11b population that were CD38 − iNOS + , the gray dots represent the CD11b population that were CD38 + iNOS − and the green dots represent the CD11b population that were double positive for both CD38 + iNOS + . For panels ( C , D ), the configuration for the colored dots is the same for the representation labeling, single, double or absent, though the proteins Arg1 and CD163 are represented rather than CD38 and iNOS.

Article Snippet: Cells were then washed once with flow cytometry staining buffer (R&D System), fixed, and permeabilized (BD Cytofix/Cytoperm, BD Bioscience) for 20 min to permit immunostaining with selected antibodies against intracellular proteins for 1 h on ice.

Techniques: Expressing, Transplantation Assay, Flow Cytometry, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Schwann Cell Transplantation Subdues the Pro-Inflammatory Innate Immune Cell Response after Spinal Cord Injury

doi: 10.3390/ijms19092550

Figure Lengend Snippet: Cytokine profile of CD11b + cells is unaltered by SC implants after SCI. Representative images of flow cytometry analysis and pie charts of CD11b population dynamics at 14 days post-injury (7 days post-transplantation) show that CD11b immune cells have unaltered expression of pro- (TNF-α, IL-1β) and anti-inflammatory (IL-4, IL-10) cytokines comparatively among SCI controls ( A – E ) and SC-transplanted groups ( F – J ). Results are expressed as mean ± SD. For panels ( A – D , F – I ) the blue dots represent the cell population that were CD11b − or did not express the selected cytokines, whereas the green dots represent the CD11b + population that expressed CD11b and the cytokine of interest (top right quadrant).

Article Snippet: Cells were then washed once with flow cytometry staining buffer (R&D System), fixed, and permeabilized (BD Cytofix/Cytoperm, BD Bioscience) for 20 min to permit immunostaining with selected antibodies against intracellular proteins for 1 h on ice.

Techniques: Flow Cytometry, Transplantation Assay, Expressing

Journal: EMBO Reports

Article Title: DJ‐1 depletion prevents immunoaging in T‐cell compartments

doi: 10.15252/embr.202153302

Figure Lengend Snippet: A Representative flow‐cytometry plots of CD44 and CD62L expression on total CD8 T cells of 45‐week‐old Dj‐1 KO and age‐ and sex‐matched WT mice. B, C Percentages of CD44 low CD62L high (Tn) (B) and CD44 high CD62L low (Tem) (C) cells among total CD8 T cells of spleen and pLNs from young and 45‐week‐old Dj‐1 KO and WT littermates (young KO, n = 5; young WT, n = 5; 45‐week‐old KO, n = 8; 45‐week‐old WT, n = 6; for 45‐week‐old mice, data pooled from 2 independent experiments). D Representative histogram overlay of PD‐1 expression among total CD8 T cells in spleen of 45‐week‐old mice (left panel) and percentages of PD‐1 + cells among total CD8 T cells (right panel). E Percentages of Ki‐67 + cells among total CD8 T cells. F Percentage of Ki‐67 + cells among splenic CD8 Tn, Tem, and Tcm in 45‐week‐old Dj‐1 KO and age‐ and sex‐matched WT mice. G Representative flow‐cytometry plots of Ki‐67 and PD‐1 among splenic CD8 Tem of 45‐week‐old mice. H IFN‐γ production in CD8 T cells of spleen and pLNs after in vitro stimulation using 50 ng/ml of PMA and 750 ng/ml of ionomycin for 5 h. I Representative flow‐cytometry plots of CD44 and CD49d among blood CD8 T cells of 60‐week‐old mice. J Percentages of Tn (top), Tvm (middle), and Tmem (bottom) among 60‐week‐old or young mice (young KO, n = 4; young WT, n = 3; 60‐week‐old KO, n = 4; and 60‐week‐old WT, n = 5). K Representative histogram of CD31 among blood CD8 T cells of 60‐week‐old mice. L CD31 MFI of total CD8 T cells (young KO, n = 4; young WT, n = 3; 60‐week‐old KO, n = 4; and 60‐week‐old WT, n = 5). Data information: SP and LN represent spleen and lymph nodes, respectively. Results represent at least four (B‐H) or two (I‐L) independent experiments. Data are mean of biological replicates ± SD. Each dot/symbol represents the measurement from one mouse. The P ‐values are determined by a two‐tailed non‐paired Student’s t ‐test. n.s. or unlabeled, not significant, * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001. Source data are available online for this figure.

Article Snippet: Spleens and peripheral lymph nodes (pLN) were collected and stored in the ice cold flow‐cytometry staining (FCM) buffer [Ca 2+ and Mg 2+ free PBS (Lonza, BE17‐516F) with 2% inactivated fetal bovine serum (FBS) and 2 mM EDTA, pH 8.0].

Techniques: Flow Cytometry, Expressing, In Vitro, Two Tailed Test

Journal: EMBO Reports

Article Title: DJ‐1 depletion prevents immunoaging in T‐cell compartments

doi: 10.15252/embr.202153302

Figure Lengend Snippet: A Expression level of CD31 among total blood or splenocyte CD4 T cells of 45‐week‐old (left) and young (right) mice. B Representative flow‐cytometry plots of CD44 and CD62L expression on total CD4 T cells of 45‐week‐old Dj‐1 KO and age‐ and sex‐matched WT mice (young KO, n = 5; young WT, n = 5; 45‐week‐old KO, n = 8; 45‐week‐old WT, n = 6; for 45‐week‐old mice, data pooled from 2 independent experiments; of note, more than one pLNs might be taken from several mice). C, D Percentages of CD44 low CD62L high (Tn) (C) and CD44 high CD62L low (Tem) (D) cells among total CD4 T cells of spleen and pLNs from young and 45‐week‐old Dj‐1 KO and WT littermates. E Representative histogram overlay of PD‐1 expression among total CD4 T cells in spleen of 45‐week‐old mice (left panel) and percentages of PD‐1 + cells among total CD4 T cells (right panel). F Representative histogram overlay of CTLA‐4 expression among total CD4 T cells in spleen of 45‐week‐old mice (left panel) and percentages of CTLA‐4 + cells among total CD4 T cells (right panel). G Percentages of Ki‐67 + cells among total CD4 T cells. H IFN‐γ production in CD4 T cells of spleen and pLNs after in vitro stimulation using 50 ng/ml of PMA and 750 ng/ml of ionomycin for 5 h. I The selected significantly enriched GO‐terms and pathways among the downregulated genes in CD4 Tconv cells from 45‐week‐old Dj‐1 KO mice versus the age‐ and gender‐matched WT littermates from microarray analysis (upper panel). Lower panel, volcano plot shows both downregulated and upregulated differentially expressed genes in splenic CD4 T cells from three 45‐week‐old Dj‐1 KO mice versus three age‐matched WT littermates. The dashed line in y axis corresponds to the value of 1.3 ( P = 0.05), while the two dashed lines in x ‐axis correspond to −1 and 1 (change fold = 2). A two‐tailed Student t ‐test was used to calculate the P values (for detailed microarray analysis method, refer to Materials and Methods). J, K Comparison of naive CD4 (Tn) mitochondrial mass (mito mass, J) and membrane potential (mito potential, K) of young and 45‐week‐old Dj‐1 KO and WT mice. L, M Comparison of CD4 Tem mitochondrial mass (mito mass, L) and membrane potential (mito potential, M) of young and 45‐week‐old Dj‐1 KO and WT mice. SP and LN represent spleen and lymph nodes, respectively. Data information: results represent at least four (B–G) and three (J–M) independent experiments. Data are mean of biological replicates ± SD. Each biological replicate indicates the measurement from one individual mouse. The P ‐values are determined by a two‐tailed un‐paired Student’s t ‐test. n.s. or unlabeled, not significant, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001.

Article Snippet: Spleens and peripheral lymph nodes (pLN) were collected and stored in the ice cold flow‐cytometry staining (FCM) buffer [Ca 2+ and Mg 2+ free PBS (Lonza, BE17‐516F) with 2% inactivated fetal bovine serum (FBS) and 2 mM EDTA, pH 8.0].

Techniques: Expressing, Flow Cytometry, In Vitro, Microarray, Two Tailed Test

Journal: EMBO Reports

Article Title: DJ‐1 depletion prevents immunoaging in T‐cell compartments

doi: 10.15252/embr.202153302

Figure Lengend Snippet: A Schematic of the experimental setup of bone marrow transplantation. A total of 10E6 of bone marrow cells from young Dj‐1 KO mice (CD45.2 + ) and WT mice (CD45.1 + ) (1:1 mix) were transferred into lethally‐irradiated young WT recipients (CD45.2 + ) by i.v. injection. Mice stably engrafted with donor cells were sacrificed for flow cytometry (FCM) analysis later. B, C Percentages of KLRG1 + CD8 T cells derived from young Dj‐1 KO and WT donor BM cells in blood (B) and spleen (C) within young WT recipients ( n = 5; blood sampled twice at both 6 and 8 weeks). D Percentages of PD‐1 + cells among total CD8 T cells derived from young Dj‐1 KO and WT BM cells in spleen within young WT recipients. E, F Percentages of CD8 Tem derived from young Dj‐1 KO and WT BM cells in blood (E) and spleen (F) within young WT recipients. G, H Ratios between CD8 Tn and Tem cells developed from CD45.1 (WT) or CD45.2 (KO) BM cells in blood (G) and spleen (H) within young WT recipients. I, J Percentages of CD8 Tn in blood (I) and spleen (J) derived from young Dj‐1 KO and WT BM cells within young WT recipients. K, L Percentage of CD8 Tcm among total CD8 T cells derived from young Dj‐1 KO and WT BM cells in blood (K) and spleen (L) of young WT recipients. M Percentages of CD8 single‐positive cells among thymus originated from 45‐week‐old Dj‐1 KO and WT BM cells within young Dj‐1 KO or WT recipients following reconstitution. N, O Percentages of CD8 CD44 high CD62L high (Tcm) cells in blood (N) and spleen (O) derived from 45‐week‐old Dj‐1 KO and WT BM cells within young Dj‐1 KO or WT recipients. Data information: Results represent two independent experiments. The P ‐values are determined by a two‐tailed paired Student’s t ‐test. n.s., not significant, * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001.

Article Snippet: Spleens and peripheral lymph nodes (pLN) were collected and stored in the ice cold flow‐cytometry staining (FCM) buffer [Ca 2+ and Mg 2+ free PBS (Lonza, BE17‐516F) with 2% inactivated fetal bovine serum (FBS) and 2 mM EDTA, pH 8.0].

Techniques: Transplantation Assay, Irradiation, Injection, Stable Transfection, Flow Cytometry, Derivative Assay, Two Tailed Test

Journal: EMBO Reports

Article Title: DJ‐1 depletion prevents immunoaging in T‐cell compartments

doi: 10.15252/embr.202153302

Figure Lengend Snippet: Schematic of the experimental setup of BM transplantation. A total of 10E6 of bone marrow cells from 45‐week‐old Dj‐1 KO mice (CD45.2 + ) and WT mice (CD45.1 + ) (1:1 mix) were transferred into lethally irradiated young Dj‐1 KO or WT recipients (CD45.2 + ) by i.v. injection. Mice stably engrafted with donor cells were sacrificed for flow cytometry (FCM) analysis later. Percentages of KLRG1 + CD8 T cells derived from 45‐week‐old Dj‐1 KO and WT BM cells within young Dj‐1 KO ( n = 4) or WT recipients ( n = 5). Percentages of KLRG1 + among CD8 Tem derived from 45‐week‐old Dj‐1 KO and WT BM cells within young Dj‐1 KO or WT recipients. Percentages of PD‐1 + population among CD8 T cells derived from 45‐week‐old Dj‐1 KO and WT BM cells within young Dj‐1 KO ( n = 4) or WT recipients ( n = 5). Percentages of CD8 + CD44 high CD62L low (Tem) cells derived from 45‐week‐old Dj‐1 KO and WT BM cells within young Dj‐1 KO or WT recipients. Ratios between blood CD8 Tn and Tem cells developed from two types of BM cells within young Dj‐1 KO or WT recipients. Percentages of CD8 + CD44 low CD62L high (Tn) cells derived from 45‐week‐old Dj‐1 KO and WT BM cells within young Dj‐1 KO or WT recipients. Schematic of the experimental setup of CD8 Tn adoptive transfer. 2.5E5 naïve CD8 T cells isolated from young or 55‐week‐old Dj‐1 KO and WT littermates were injected into Rag‐1 deficient mice by i.v. injection. 6 weeks later, mice were sacrificed for FCM (flow cytometry) analysis. Representative flow‐cytometry plots of KLRG1 + population among total CD8 T following adoptive transfer of CD8 Tn from 55‐week‐old mice. Percentages of KLRG1 + population among total CD8 T cells (55‐week‐old KO, n = 4; 55‐week‐old WT, n = 5; young KO, n = 4; and young WT, n = 4). Percentages of KLRG1 + population among CD8 Tem cells. Percentages of KLRG1 + PD‐1 + population among total CD8 T cells (55‐week‐old KO, n = 4; 55‐week‐old WT, n = 5; young KO, n = 4; and young WT, n = 4). Percentages of KLRG1 + PD‐1 + population among CD8 Tem cells. Data information: Results from BM transfer and adoptive transfer of CD8 Tn represent two independent experiments. Data are mean ± SD. The P ‐values are determined by a two‐tailed paired (B–G) or non‐paired (J–M) Student’s t ‐test. n.s. or unlabeled, not significant, * P ≤ 0.05 and ** P ≤ 0.01.

Article Snippet: Spleens and peripheral lymph nodes (pLN) were collected and stored in the ice cold flow‐cytometry staining (FCM) buffer [Ca 2+ and Mg 2+ free PBS (Lonza, BE17‐516F) with 2% inactivated fetal bovine serum (FBS) and 2 mM EDTA, pH 8.0].

Techniques: Transplantation Assay, Irradiation, Injection, Stable Transfection, Flow Cytometry, Derivative Assay, Adoptive Transfer Assay, Isolation, Two Tailed Test

Journal: EMBO Reports

Article Title: DJ‐1 depletion prevents immunoaging in T‐cell compartments

doi: 10.15252/embr.202153302

Figure Lengend Snippet: Representative flow cytometry data of CD69 and Celltrace violet (CTV) staining on gated living CD8 T cells. The purified CD8 Tn were isolated from young mice and stimulated by different doses of anti‐CD3 ab for 72 h. Enlarged number representing the percentage of the corresponding gate. The percentages of proliferating cells among living CD8 singlets (CD8 Tn were from young mice). The percentages of CD69 + cells among living CD8 T singlets (CD8 Tn were from young mice). Representative flow cytometry data of CD69 and CTV staining on gated living CD8 T cells. The purified CD8 Tn were isolated from 45‐week‐old mice and stimulated by different doses of anti‐CD3 ab for 72 h. Enlarged number representing the percentage of the corresponding gate. The percentages of proliferating cells among living CD8 singlets (CD8 Tn were from 45‐week‐old mice). The percentages of CD69 + cells among living CD8 T singlets (CD8 Tn were from 45‐week‐old mice). Data information: Results represent at least two independent experiments (A‐F). Data are mean ± SD. All the CD8 Tn cells were first pooled from 3 to 4 mice of the same group before exposing to different doses of anti‐CD3 ab. The error bar (SD) here essentially refers to technical replicates. The P ‐values are determined by a two‐tailed non‐paired (B‐C, E‐F) Student’s t ‐test. Unlabeled, not significant, * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001.

Article Snippet: Spleens and peripheral lymph nodes (pLN) were collected and stored in the ice cold flow‐cytometry staining (FCM) buffer [Ca 2+ and Mg 2+ free PBS (Lonza, BE17‐516F) with 2% inactivated fetal bovine serum (FBS) and 2 mM EDTA, pH 8.0].

Techniques: Flow Cytometry, Staining, Purification, Isolation, Two Tailed Test

Journal: Advanced Science

Article Title: NAD + Metabolism Reprogramming Drives SIRT1‐Dependent Deacetylation Inducing PD‐L1 Nuclear Localization in Cervical Cancer

doi: 10.1002/advs.202412109

Figure Lengend Snippet: Comparison Between SIRT1 and HDACs in Regulating PD‐L1. A) Flow cytometry of membrane PD‐L1 expression after supplying with LBH589 in HeLa. B) Flow cytometry of membrane PD‐L1 expression after supplying LBH589 alone or in combination with ITSA‐1 (50 µ m ) pretreatment in HeLa. C) Flow cytometry of membrane PD‐L1 expression after supplying with EX527 in HeLa. D, E) Flow cytometry of membrane PD‐L1 expression after supplying with NAD + or NMN alone or in combination with EX527 pretreatment in HeLa. F, G) qPCR and Western blot analysis of overall expression of PD‐L1 after supplying with SIRT1 activator SRT1720 (160 n m ). H, I) qPCR and Western blot analysis of overall expression of PD‐L1 after supplying with SIRT1 substrate NAD + . J–L) qPCR and Western blot analysis of overall expression of PD‐L1 after supplying with EX527. M, N) qPCR and Western blot analysis of overall expression of PD‐L1 after supplying with LBH589. O) Scheme representing the experimental procedure. NMN (150mg kg −1 ) was administered daily with water. FK866 (20mg kg −1 ) and αPD‐L1(3mg kg −1 ) are injected intraperitoneally twice a week. P–S) Tumor size (P), tumor growth curves (Q), tumor volume (R), and body weight (S) of C57BL/6 mice injected subcutaneously with U14 cells (n = 4). Data were represented as mean ± SD. Two‐tailed unpaired Student's t‐tests. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Resuspend the cells in 50 μL of Flow Cytometry Staining Buffer (Elabscience).

Techniques: Comparison, Flow Cytometry, Membrane, Expressing, Western Blot, Injection, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Molecular Characterization of Gastric Epithelial Cells Using Flow Cytometry

doi: 10.3390/ijms19041096

Figure Lengend Snippet: Gating strategy for analyzing gastric epithelial cells by flow cytometry. ( A ) A gate based of forward area and side scatter area is first established, these cells are then put through a forward scatter area and forward scatter height gate to identify single cells, finally, 7-AAD is used to separate live cells from dead/dying cells; ( B ) Representative flow plots of live single cells from healthy BALB/c and TxA23 mice stained with an epithelial cell marker (EpCAM) and an immune cell marker (CD45). Immune cells are undetectable in the gastric mucosa control mice, and present in TxA23 mice that have autoimmune gastritis; ( C ) Representative immunocytochemistry of glands isolated from a 2 month old BALB/c mouse and stained with hoechst (blue), parietal cell marker Dolichous bifluorous agglutinin (DBA, green), and anti-EpCAM (red) with a high magnification inset in yellow showing an individual gland; ( D ) A representative flow cytometry plot of live single cells from a BALB/c mouse stained with anti-EpCAM and DBA demonstrating a subset of EpCAM + DBA + parietal cells.

Article Snippet: Following Dispase digestion, single cells were washed twice using flow cytometry staining buffer supplemented with 20 mM EDTA (Promega, Madison, WI, USA, V4231) and counted using Trypan blue exclusion.

Techniques: Flow Cytometry, Staining, Marker, Control, Immunocytochemistry, Isolation