Journal: Frontiers in Cell and Developmental Biology

Article Title: Human placenta-derived mesenchymal stem cells stimulate neuronal regeneration by promoting axon growth and restoring neuronal activity

doi: 10.3389/fcell.2023.1328261

Figure Lengend Snippet: Characterization of hPMSCs. (A) Morphology at passage 3. Both normoxic and hypoxic cultures show highly homogeneous spindle-like shape. Scale bar: 500 µm (B) Proliferation capacity. Cumulative population doublings of hPMSCs grown under hypoxia (square) are higher than cultures grown in normoxia (dot). (C) Flow cytometry shows specific hMSCs marker expression pattern: positive for CD105, CD44, CD90, CD73 and negative for CD14, CD19, CD34, CD45 and HLA-DR. (D) Osteogenic differentiation was confirmed by Alizarin red staining of calcium deposits. (E) Adipogenesis differentiation was followed by Oil Red O staining of lipids vacuoles (black arrows indicate lipid droplets). (F) Alcian Blue staining of proteoglycans demonstrated chondrogenesis differentiation. Scale bar: 100 μm. Two-way ANOVA and post hoc Sidak test for multiple comparisons between means (** p ≤ 0.005; * p ≤ 0.05).

Article Snippet: Antibodies against the following human antigens were used: CD105-FITC (Miltenyi Biotect, Bergisch Gladbach, Germany, cat# 130-112-327, 1:50), CD90-FITC (Miltenyi Biotec, cat# 130-114-901, 1:50), CD44-VioBlue (Miltenyi Biotec, cat# 130-113-906, 1:50), CD73-APC (Miltenyi Biotec, cat# 130-111-909, 1:50), MSC Phenotyping Cocktail-PE (CD34, CD14, CD19, CD45, Miltenyi Biotec cat# 130-125-285, dilution according to the manufacturer’s instructions).

Techniques: Flow Cytometry, Marker, Expressing, Staining

Journal: Cell reports

Article Title: The aryl hydrocarbon receptor cell intrinsically promotes resident memory CD8 + T cell differentiation and function

doi: 10.1016/j.celrep.2022.111963

Figure Lengend Snippet: (A and B) Flow cytometry analysis of Ahr expression (GFP) in CD8 + T cells of Ahr +/+ and Ahr dCAIR/+ mice was performed. Histogram plot of GFP( Ahr ) in CD8 + T cells isolated from spleen (Sp), peripheral lymph node (pLN), mesenteric lymph node (mLN), small intestine lamina propria (LPL), or intraepithelial lymphocytes (IEL) (A). Quantification of ΔgMFI in Ahr dCAIR/+ compared with GFP-negative Ahr +/+ mice (B). (C and D) Flow cytometry quantification of ΔgMFI of Ahr -GFP in different CD8 + T cell populations isolated from LPL including CD44 − CD62L + (T naive ), CD127 − KLRG1 − (T EE ), CD127 − KLRG1 + (T SLE ), CD127 + KLRG1 − (T MPE ) (C), and CD44 + CD62L + (T CM ), CD44 + CD62L − (T EM ), and CD69 + CD103 + (T RM ) (D). Data are shown as mean ± SEM (n = 3 mice per group). Data are representative of two independent experiments. See also .

Article Snippet: Anti-human CD8 – APC-Cyanine7 (clone SK1) , TONBO biosciences , Cat# 25-0087-T025; RRID: AB_2848136.

Techniques: Flow Cytometry, Expressing, Isolation

Journal: Cell reports

Article Title: The aryl hydrocarbon receptor cell intrinsically promotes resident memory CD8 + T cell differentiation and function

doi: 10.1016/j.celrep.2022.111963

Figure Lengend Snippet: (A–E and H) RNA-seq analysis of IEL resident CD8 + T cells isolated from Ahr f/f and Ahr f/f Cd8 cre mice and statistics (q values shown) were calculated via DESeq2 differential expression analysis, and FPKM values were quantified using RSEM. MA plot highlighting genes upregulated (red) and downregulated (blue) in Ahr f/f Cd8 cre compared with Ahr f/f (A). Gene set enrichment analysis of resident and circulating core gene signatures (B). Heatmap depicting (fold change >1.5) genes enriched in respective signatures (C). FPKM expression values for transcriptional regulators (D) and secreted factors associated with cell function (E). (F and G) Flow cytometry quantification of granzyme B protein levels of IEL resident CD8 + T cells isolated from Ahr f/f and Ahr f/f Cd8 cre mice. The percentages (F) and total cell number (n = 5 mice per group) (G) are shown. Data are representative of two independent experiments. (H) FPKM expression values for proliferation, cell cycle, and apoptosis genes. (I–K) Naive CD8 + T cells were isolated from Ahr +/+ and Ahr − / − mice and then subjected to in vitro T RM -like differentiation culture conditions. RNA was isolated for qRT-PCR expression analysis of Ahrr (I). Flow cytometry quantification of CD69 + CD103 + in vitro T RM -like cells (J) and CD44 + CD62L + in vitro T CM -like population frequency (K). Data are shown as mean ± SEM (n = 3 mice per group). Data are representative of three independent experiments. See also , , and .

Article Snippet: Anti-human CD8 – APC-Cyanine7 (clone SK1) , TONBO biosciences , Cat# 25-0087-T025; RRID: AB_2848136.

Techniques: RNA Sequencing, Isolation, Quantitative Proteomics, Expressing, Cell Function Assay, Flow Cytometry, In Vitro, Quantitative RT-PCR

Journal: Cell reports

Article Title: The aryl hydrocarbon receptor cell intrinsically promotes resident memory CD8 + T cell differentiation and function

doi: 10.1016/j.celrep.2022.111963

Figure Lengend Snippet: (A and B) ChIP-seq of Ahr was performed using in vitro T RM -like CD8 + T cells. Analysis of Ahr binding was performed, and pie chart of peak annotation (A) as well as top seven enriched transcription factor motifs (B) are shown. (C and D) Binding and Expression Target Analysis (BETA) was performed to integrate RNA-seq and ChIP-seq data. Visualization of transcription factor activating/repressive function prediction (C) and rank product volcano plot depicting top direct target candidates (D). (E and F) Ahr − / − in vitro T RM -like CD8 + T cells transduced with retroviral constructs encoding MIG-EV (empty vector), MIG-Ahr, MIG-Y9A, or MIG-DbHLH. The cells were treated with DMSO or FICZ on day 3. On day 5, CD69 and CD103 expression was analyzed by flow cytometry (E), and RNA was isolated for qRT-PCR expression analysis of Ahr direct target gene Ahrr (F). Data are shown as mean ± SEM (n = 3 technical replicates per group). Data are representative of two independent experiments. (G) Flow cytometry analysis of CD69 and CD103 expression in Ahr − / − in vitro T RM -like CD8 + T cells transduced with retroviral constructs encoding MIG-EV, MIG-Ahr, hCD2-EV, or hCD2-Blimp1. Data are representative of two independent experiments. See also .

Article Snippet: Anti-human CD8 – APC-Cyanine7 (clone SK1) , TONBO biosciences , Cat# 25-0087-T025; RRID: AB_2848136.

Techniques: ChIP-sequencing, In Vitro, Binding Assay, Expressing, RNA Sequencing, Transduction, Retroviral, Construct, Plasmid Preparation, Flow Cytometry, Isolation, Quantitative RT-PCR

Journal: Cell reports

Article Title: The aryl hydrocarbon receptor cell intrinsically promotes resident memory CD8 + T cell differentiation and function

doi: 10.1016/j.celrep.2022.111963

Figure Lengend Snippet: (A–F) Analysis of antigen-specific (OTI) CD8 + T cell response in the IEL during oral L . m .-OVA infection was performed as depicted in the schematic (A). Flow cytometry quantification of the percentage of Ahr +/+ vs. Ahr − / − OTI cells on day 9, 20, and 34 post infection (B). Flow cytometry quantification of memory precursor populations based on expression of CD127 and KLRG1 (C) as well as CD69 and CD103 (D) in Ahr +/+ vs. Ahr − / − OTI cells present in the IEL on day 9 post infection. Flow cytometry analysis of CD45.1 and GzmB gated on OTI cells depicting CD45 . 1 + Ahr +/+ (CD45.1/.2) and CD45 . 1 − Ahr − / − (CD45.2/.2) OTI cells production of granzyme B on day 34 post infection (E). Quantification of granzyme B + OTI T cells in the mice of indicated genotypes (F). (G–J) L . m .-OVA re-infection was performed and Ahr +/+ vs. Ahr − / − OTI IEL resident CD8 + T cells analyzed on day 3 post re-infection. Flow cytometry analysis of CD45.1 and CD45.2 depicting percentage of Ahr +/+ (CD45.1/.2) and Ahr − / − (CD45.2/.2) OTI IEL resident CD8 + T cells as well as (G) quantification of percentage are shown (H). Flow cytometry analysis (I) and quantification (J) of granzyme B production in analyzed cells. Data are compiled from two independent experiments and shown as mean ± SEM (n = 3–6 replicates per group). See also .

Article Snippet: Anti-human CD8 – APC-Cyanine7 (clone SK1) , TONBO biosciences , Cat# 25-0087-T025; RRID: AB_2848136.

Techniques: Infection, Flow Cytometry, Expressing

Journal: Cell reports

Article Title: The aryl hydrocarbon receptor cell intrinsically promotes resident memory CD8 + T cell differentiation and function

doi: 10.1016/j.celrep.2022.111963

Figure Lengend Snippet: (A–D) scRNA-seq analysis of Ahr +/+ (n = 2) vs. Ahr − / − (n = 2) TIL CD8 + T cells was performed. UMAP dimensionality reduction and cluster visualization (left) as well as pie chart frequency depiction (right) color-coded to represent cluster ID (A). Pseudotime visualization (B) and quantification (C). Differential gene expression depicted via color intensity as average expression and circle size as percent expressed in CD8 + T cells (D). (E–H) Ahr f/f and Ahr f/f Cd8 cre mice were inoculated subcutaneously with B16F10 mouse melanoma and tumor size monitored (E). At endpoint, tumor weight was quantified (F), and TILs were isolated for flow cytometry analysis. Pie chart visualization depicting polyfunctionality of TIL CD8 + T cells in mice with indicated genotypes (G). Polyfunctionality score quantification (triple-positive plus double-positive minus triple-negative divided by total cells) of TIL CD8 + T cells isolated from Ahr f/f and Ahr f/f Cd8 cre tumor-bearing mice (H). Data are compiled from two independent experiments and are shown as mean ± SEM (n = 6 to 7 replicates per group). See also .

Article Snippet: Anti-human CD8 – APC-Cyanine7 (clone SK1) , TONBO biosciences , Cat# 25-0087-T025; RRID: AB_2848136.

Techniques: Gene Expression, Expressing, Isolation, Flow Cytometry

Journal: Cell reports

Article Title: The aryl hydrocarbon receptor cell intrinsically promotes resident memory CD8 + T cell differentiation and function

doi: 10.1016/j.celrep.2022.111963

Figure Lengend Snippet: (A–C) Human peripheral blood (PBMC) vs. IEL CD8 + T cells were analyzed via flow cytometry. Staining of CD45RA and CD45RO (top), CD103 and CD69 (bottom) (A), as well as T-BET and AHR (B) in tissue fractions as indicated in the figure. Quantification of AHR protein levels (gMFI) in human PBMC, LPL, and IEL CD8 + T cells (C). Data are compiled from three independent experiments and shown as mean ± SEM (n = 4 replicates per group). (D–G) Human naive CD8 + T cells were isolated from PBMCs and then subjected to in vitro T RM -like differentiation culture conditions. The cells were given differentiation cytokines and treated with DMSO (control), FICZ, or CH223191 on day 2. The assay was collected on day 4, and flow cytometry quantification of CD103 expression was performed (D). Data are compiled from three independent experiments and are shown as mean ± SEM (n = 4 replicates per group). RNA was isolated for qRT-PCR expression analysis of AHR (E), AHR direct target gene AHRR (F), and GZMB (G). Data are shown as mean ± SEM (n = 3 technical replicates per group). Data are representative of three independent experiments.

Article Snippet: Anti-human CD8 – APC-Cyanine7 (clone SK1) , TONBO biosciences , Cat# 25-0087-T025; RRID: AB_2848136.

Techniques: Flow Cytometry, Staining, Isolation, In Vitro, Control, Expressing, Quantitative RT-PCR

Journal: Cell reports

Article Title: The aryl hydrocarbon receptor cell intrinsically promotes resident memory CD8 + T cell differentiation and function

doi: 10.1016/j.celrep.2022.111963

Figure Lengend Snippet:

Article Snippet: Anti-human CD8 – APC-Cyanine7 (clone SK1) , TONBO biosciences , Cat# 25-0087-T025; RRID: AB_2848136.

Techniques: Virus, Isolation, Recombinant, SYBR Green Assay, DNA Library Preparation, Reverse Transcription, Staining, Transgenic Assay, Plasmid Preparation, Software