Journal: International Journal of Molecular Sciences

Article Title: Reduced Dendritic Cells Expressing CD200R1 in Children with Inflammatory Bowel Disease: Correlation with Th17 and Regulatory T Cells

doi: 10.3390/ijms161226143

Figure Lengend Snippet: Representative flow cytometry data to illustrate the gating strategy for FACS analysis of DC subsets. ( A ) Dendritic cell HLA-DR + Lin − cells were gated ( R1 ), and myeloid CD11c + mDCs ( R2 ), plasmacytoid CD123 + pDCs ( R3 ) were determined as a percentage of the total PBMC ( B ). Dot plots to illustrate the expression of CD200 and CD200R1 on the myeloid dendritic cell (mDC) subset of healthy controls and IBD patients ( C , D ) and on and plasmacytoid DCs (pDCs) of healthy controls and IBD patients ( E , F ).

Article Snippet: For regulatory T cell staining, 1 × 10 6 cells were resuspended in 100 μL flow cytometry staining buffer (R&D Systems, Minneapolis, MN, USA).

Techniques: Flow Cytometry, Expressing

Journal: Science advances

Article Title: Profiling of mature-stage human breast milk cells identifies six unique lactocyte subpopulations.

doi: 10.1126/sciadv.abm6865

Figure Lengend Snippet: Fig. 1. Maternal cells in breast milk were identified by protein, gene, and transcriptome analyses. Mature-stage breast milk from healthy donors was assessed for expression of cell marker proteins (e.g., EpCAM and CD45) in freshly isolated samples by flow cytometry. Then, RNA was isolated and used to conduct complementary gene expression analysis by RT-qPCR. Single-cell transcriptomics added depth to the analysis and defined the lactocyte population in breast milk.

Article Snippet: Fixable Yellow samples were subsequently fixed using Flow Cytometry Fixation Buffer (R&D Systems), centrifuged, and resuspended in 1% FBS in PBS.

Techniques: Expressing, Marker, Isolation, Flow Cytometry, Gene Expression, Quantitative RT-PCR, Single-cell Transcriptomics

Journal: Science advances

Article Title: Profiling of mature-stage human breast milk cells identifies six unique lactocyte subpopulations.

doi: 10.1126/sciadv.abm6865

Figure Lengend Snippet: Fig. 2. Mature-stage breast milk from donors contained epithelial lactocytes and a smaller population of immune cells. (A) Flow cytometry quantified expression of cell-specific markers: EpCAM+ (epithelial cells), CD45+ (immune cells), CK18+ (lactocytes), and VIM+ (mesenchymal cells). These data suggest that the CK18 antibody bounds poorly to lactocytes and likely underestimates true lactocyte percentages. FITC, fluorescein isothiocyanate. (B) The relative cellular composition in breast milk was affected by the health status of the mother and infant. Representative density plots are shown of EpCAM versus CD45 cell populations in milk from healthy donors (n = 10), donors with sick infants (n = 1), or donors with recent mastitis (n = 3). (C) Flow cytometry analysis of protein markers in all health statuses (n = 14) and the breakdown of (D) CD45+ immune cells and (E) VIM+ mesenchymal cells in different health statuses. (F) mRNA expression of gene markers corresponding to proteins in (C) indicates the presence of epithelial lactocytes (n = 6 to 7). Data are shown as means ± SEM. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns, not significant.

Article Snippet: Fixable Yellow samples were subsequently fixed using Flow Cytometry Fixation Buffer (R&D Systems), centrifuged, and resuspended in 1% FBS in PBS.

Techniques: Flow Cytometry, Expressing, Comparison

Journal: Science advances

Article Title: Profiling of mature-stage human breast milk cells identifies six unique lactocyte subpopulations.

doi: 10.1126/sciadv.abm6865

Figure Lengend Snippet: Fig. 3. Stem-like transcription factors were expressed in 5% of breast milk cells. Four stem-like transcription factors were assessed in breast milk epithelial cells. (A) From flow cytometry analysis, representative histograms of SOX2+, TRA-1-60+, NANOG+, and SSEA4+ stained and unstained samples are shown. (B) Flow cytometry analysis of EpCAM+ cells (n = 15) indicated high expression of SOX2 and TRA-1-60 with moderate to low expression of NANOG and SSEA4, respectively. (C) mRNA expression relative to a housekeeping gene was quantified for the genes corresponding to the proteins in (B) (n = 7). (D) Flow cytometry analysis of EpCAM+ cells and their increasing stemness based on positive expression of multiple stem-like markers (n = 15). Data are means ± SEM. One-way ANOVA with Tukey’s multiple comparison test; *P < 0.05; ****P < 0.0001.

Article Snippet: Fixable Yellow samples were subsequently fixed using Flow Cytometry Fixation Buffer (R&D Systems), centrifuged, and resuspended in 1% FBS in PBS.

Techniques: Flow Cytometry, Staining, Expressing, Comparison

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: 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