Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Coexpression of TIGIT and FCRL3 Identifies Helios + Human Memory Regulatory T Cells

doi: 10.4049/jimmunol.1401803

Figure Lengend Snippet: Gene expression analysis of suppressive versus nonsuppressive FOXP3+ clones. Illumina BeadChip analysis was used for total mRNA prepared from individual clones representing three populations: 1) suppressive and 2) nonsuppressive FOXP3+ clones generated from CD4+CD25high cells, and 3) control FOXP3− clones generated from CD4+CD25− cells. (A) Correlation of suppressive potency with FOXP3 expression levels in primary FOXP3+ and FOXP3− (CD25−) clones generated from three healthy donors. The dotted lines represent the cutoffs for FOXP3 mean fluorescence intensity (MFI) (x-axis) and percentage suppression (y-axis) determined through the overall FOXP3 MFI and suppressive capacity of the control FOXP3− clones generated from CD4+CD25− cells. (B) Schematic illustrating the process of selection of representative clones. (C) Scatter plot showing normalized gene expression level in suppressive versus nonsuppressive clones in the resting state. (D) Heat map comparing variations in the expression of selected Treg- and Tconv-associated genes relative to the median mRNA levels across the three subsets. (E) Relative mRNA expression levels of TIGIT, FCRL3, and Helios in the three functional categories. Representative clones were derived from two healthy donors, and two to three similar clones/subset/donor were pooled to obtain sufficient mRNA.

Article Snippet: To investigate the activation-induced modulation of marker expression, CD4 + CD25 − TIGIT − FCRL3 − cells from healthy PBMCs were FACS sorted, labeled with CFSE, and activated for 4–6 d in vitro with anti-CD3/anti-CD28–coated beads (Miltenyi Biotec) at a ratio of two beads/one cell.

Techniques: Gene Expression, Clone Assay, Generated, Control, Expressing, Fluorescence, Selection, Functional Assay, Derivative Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Coexpression of TIGIT and FCRL3 Identifies Helios + Human Memory Regulatory T Cells

doi: 10.4049/jimmunol.1401803

Figure Lengend Snippet: FPSN clones comprise majorly Helios− clones and display a reduced FCRL3 expression. Primary Treg and Tconv clones were generated by single cell cloning of FACS-sorted CD25high and CD25− cells of four healthy individuals. Marker expression analysis was performed immediately after the harvest on days 22–24, and 4-d CFSE-based suppression assays were carried out using allogeneic CD4+CD25− cells as responders at a 1:1 Treg/Tresp ratio in the presence of irradiated PBMCs and anti-CD3 (30 ng/ml). Suppression was measured relative to the division index of the unsuppressed Tresp-alone control. Shown are the expression levels of FOXP3, Helios, TIGIT, and FCRL3 in FPSP, FPSN, and FNSN clones immediately after harvest. Statistical analysis was done with the one-way ANOVA followed by a Tukey posttest. **p ≤ 0.01, ***p ≤ 0.001. n.s., not significant.

Article Snippet: To investigate the activation-induced modulation of marker expression, CD4 + CD25 − TIGIT − FCRL3 − cells from healthy PBMCs were FACS sorted, labeled with CFSE, and activated for 4–6 d in vitro with anti-CD3/anti-CD28–coated beads (Miltenyi Biotec) at a ratio of two beads/one cell.

Techniques: Clone Assay, Expressing, Generated, Cloning, Marker, Irradiation, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Coexpression of TIGIT and FCRL3 Identifies Helios + Human Memory Regulatory T Cells

doi: 10.4049/jimmunol.1401803

Figure Lengend Snippet: The combined expression of TIGIT and FCRL3 discriminates between Helios+ and Helios− subsets in memory FOXP3+ cells. (A–C) PBMCs from healthy subjects were analyzed ex vivo by flow cytometry. (A) Expression of TIGIT, FCRL3, and Helios on naive and memory CD4+FOXP3+ populations. (B) Representative plots showing the correlation of TIGIT and FCRL3 expression with Helios expression in naive versus memory CD4+ FOXP3+ cells. (C) Combined analysis of 11 healthy individuals showing the application of TIGIT and FCRL3 in the identification Helios+ and Helios− subsets within memory FOXP3+ cells. (D) Applicability of TIGIT/FCRL3 combination in discriminating Helios subsets in FOXP3+ clones generated from seven healthy donors (n = 299 clones).

Article Snippet: To investigate the activation-induced modulation of marker expression, CD4 + CD25 − TIGIT − FCRL3 − cells from healthy PBMCs were FACS sorted, labeled with CFSE, and activated for 4–6 d in vitro with anti-CD3/anti-CD28–coated beads (Miltenyi Biotec) at a ratio of two beads/one cell.

Techniques: Expressing, Ex Vivo, Flow Cytometry, Clone Assay, Generated

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Coexpression of TIGIT and FCRL3 Identifies Helios + Human Memory Regulatory T Cells

doi: 10.4049/jimmunol.1401803

Figure Lengend Snippet: TIGIT/FCRL3 combination provides a reliable surface marker for the isolation of Helios+ and Helios− memory Treg cells. PBMCs from 11 healthy subjects were analyzed ex vivo by flow cytometry. (A) Representative FACS plots showing the application of the TIGIT/FCRL3 marker combination in distinguishing Helios subsets within memory CD4+CD25+CD127low cells. (B) Identification of Helios subsets by TIGIT/FCRL3 surface markers is precisely reproducible in healthy samples with a wide range of Helios expression. Shown is the frequency of Helios+ (top) and FOXP3+ (bottom) cells in Treg populations gated using the conventional markers (CD25+CD127low; referred to as Total) compared with further gating using different combinations of TIGIT and FCRL3. (C) The TIGIT/FCRL3 combination allows the identification of consistently enriched FOXP3+Helios+ populations with less stringent gating on CD25. Shown is the frequency of FOXP3+ and Helios+ cells in TIGIT+FCRL3+ cells obtained from variably stringent CD25+ gates on memory CD4+ cells.

Article Snippet: To investigate the activation-induced modulation of marker expression, CD4 + CD25 − TIGIT − FCRL3 − cells from healthy PBMCs were FACS sorted, labeled with CFSE, and activated for 4–6 d in vitro with anti-CD3/anti-CD28–coated beads (Miltenyi Biotec) at a ratio of two beads/one cell.

Techniques: Marker, Isolation, Ex Vivo, Flow Cytometry, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Coexpression of TIGIT and FCRL3 Identifies Helios + Human Memory Regulatory T Cells

doi: 10.4049/jimmunol.1401803

Figure Lengend Snippet: The TIGIT/FCRL3 marker expression can reliably identify Helios+ and Helios− Treg cell subsets in inflammatory contexts. (A) FACS-sorted TIGIT−FCRL3− memory CD4+CD25− cells from PBMCs of a healthy donor were labeled with CFSE and activated in vitro with anti-CD3/anti-CD28–coated beads at a ratio of two beads/one cell for 5 d. Shown are representative FACS plots and (B) the kinetics of activation-induced marker upregulation in three separate experiments on three healthy individuals. (C) Total CD4+CD25−TIGIT−FCRL3− cells from PBMCs activated in vitro with anti-CD3/anti-CD28–coated beads at a ratio of two beads/one cell with or without recombinant human IL-2 in the presence of irradiated autologous feeders for 4 d. Shown is the expression of FCRL3 and Helios on the activated CD4+CD25−TIGIT−FCRL3− cells compared with FACS-sorted CD4+CD25+CD127lowTIGIT+FCRL3+ cells plated in parallel. (D) Whole PBMCs from a healthy individual were stimulated with anti-CD3/anti-CD28–coated beads at a ratio of two beads/one cell for 72 h. Representative FACS plots from similar experiments on three healthy donors are shown.

Article Snippet: To investigate the activation-induced modulation of marker expression, CD4 + CD25 − TIGIT − FCRL3 − cells from healthy PBMCs were FACS sorted, labeled with CFSE, and activated for 4–6 d in vitro with anti-CD3/anti-CD28–coated beads (Miltenyi Biotec) at a ratio of two beads/one cell.

Techniques: Marker, Expressing, Labeling, In Vitro, Activation Assay, Recombinant, Irradiation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Coexpression of TIGIT and FCRL3 Identifies Helios + Human Memory Regulatory T Cells

doi: 10.4049/jimmunol.1401803

Figure Lengend Snippet: An increased frequency of nonsuppressive clones is found within the FOXP3+Helios− subset. The suppressive potency of Helios+ and Helios− Treg cells was compared ex vivo (A and B) and in primary clones (C–E). For ex vivo suppression assays, CD4+CD25+CD127lowTIGIT+FCRL3+ and CD4+CD25+CD127lowTIGIT−FCRL3− cells were FACS sorted and tested for the capacity to suppress the proliferation of CFSE-labeled CD4+CD25− Tresp cells stimulated with soluble anti-CD3 and irradiated PBMCs for 4 d. (A) Representative CFSE dilution histograms showing suppression of Tresp cells. (B) Combined suppression analysis from three different experiments using cells from three different healthy individuals. (C–E) Primary Treg and Tconv clones were generated by single-cell cloning of FACS-sorted CD25high and CD25− cells of seven healthy individuals. Marker expression was performed immediately after the harvest on days 22–24, and 4-d CFSE-based suppression assays were carried out using allogeneic CD4+CD25− cells as responders at a 1:1 Treg/Tresp ratio in the presence of irradiated PBMCs and anti-CD3 (30 ng/ml). (C) The expression of CD25, FOXP3, and Helios in representative clones at harvest. (D and E) Suppressive potency of FOXP3+Helios+ (n = 196 clones), FOXP3+Helios− (n = 103 clones), and FOXP3− (n = 59 clones). Suppression was measured relative to the division index of the unsuppressed Tresp-alone control. Statistical analysis was done with the one-way ANOVA followed by a Tukey posttest. ***p ≤ 0.001.

Article Snippet: To investigate the activation-induced modulation of marker expression, CD4 + CD25 − TIGIT − FCRL3 − cells from healthy PBMCs were FACS sorted, labeled with CFSE, and activated for 4–6 d in vitro with anti-CD3/anti-CD28–coated beads (Miltenyi Biotec) at a ratio of two beads/one cell.

Techniques: Clone Assay, Ex Vivo, Labeling, Irradiation, Generated, Cloning, Marker, Expressing, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Coexpression of TIGIT and FCRL3 Identifies Helios + Human Memory Regulatory T Cells

doi: 10.4049/jimmunol.1401803

Figure Lengend Snippet: Inflammatory cytokine production in FOXP3+ cells is restricted to the Helios− fraction. (A) PBMCs were incubated ex vivo with PMA (25 ng/ml), ionomycin (1 μg/ml), and GolgiStop for 4 h, followed by intracellular cytokine staining. Shown are representative flow cytometry plots (A) and the frequency of cytokine-producing cells in the indicated subsets (B) analyzed from 11 different healthy samples. (C) Cytokine production in healthy FACS-sorted TIGIT+FCRL3+ versus TIGIT−FCRL3− Treg cells (CD4+CD45RA−CD25+CD127low). Statistical analysis was done with the one-way ANOVA followed by a Tukey posttest (B) or with the Student t test (C). *p ≤ 0.05, ***p ≤ 0.001. n.s., not significant.

Article Snippet: To investigate the activation-induced modulation of marker expression, CD4 + CD25 − TIGIT − FCRL3 − cells from healthy PBMCs were FACS sorted, labeled with CFSE, and activated for 4–6 d in vitro with anti-CD3/anti-CD28–coated beads (Miltenyi Biotec) at a ratio of two beads/one cell.

Techniques: Incubation, Ex Vivo, Staining, Flow Cytometry

Journal:

Article Title: Canarypox Virus-Induced Maturation of Dendritic Cells Is Mediated by Apoptotic Cell Death and Tumor Necrosis Factor Alpha Secretion

doi:

Figure Lengend Snippet: Maturation of DCs in vCP172-infected cultures. (A) Immature human DCs were infected with vCP172 (MOI of 10). Infected (+) and uninfected (−) DCs were cultured for 4 days. After culture, the cells were harvested and monitored for the expression of CD25-, CD83-, and CD86-PE (log PE y axes) versus HLA-DR–FITC (x axes). The percentage of large cells expressing CD25 and CD83 above the isotype control are indicated. (B) Immature rhesus macaque DCs were infected with vCP172 (+) (MOI of 10) or not (−) and cultured for 1 to 3 days before being harvested and analyzed. FACS analysis was performed on cells stained with FITC–anti-HLA-DR versus PE-immunoglobulin, -anti-CD25, -CD80, -CD83, or -CD86. Similar data were obtained from more than five experiments with human DCs and three different monkey donors.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated monoclonal Abs (MAbs) against human major histocompatibility complex (MHC) class II (anti-HLA-DR-FITC) (Becton Dickinson Immunocytometry Systems [BDIS], San Jose, Calif.) were used in combination with phycoerythrin (PE)-conjugated MAbs against human CD25 (BDIS), CD80 (BDIS), CD83 (Coulter Corp., Miami, Fla.), and CD86 (PharMingen, San Diego, Calif.).

Techniques: Infection, Cell Culture, Expressing, Staining

Journal:

Article Title: Canarypox Virus-Induced Maturation of Dendritic Cells Is Mediated by Apoptotic Cell Death and Tumor Necrosis Factor Alpha Secretion

doi:

Figure Lengend Snippet: Induction of maturation is stimulated by viable canarypox virus. (A) Immature human DCs were infected with an MOI of 10 of either vCP180 or the parental strain (ALVAC) or left uninfected (medium). CD25 surface expression by large HLA-DR-positive cells was assessed 4 days after infection (CD25-PE, y axes; HLA-DR–FITC, x axes). The percentage of CD25-positive cells (above isotype control) are indicated in each panel (highlighted by arrowheads). (B) Immature human DCs that had been infected with vCP180 3 to 4 days earlier were sorted into CD25-negative (CD25 neg.) and CD25-positive (CD25 pos.) fractions. Each fraction was then immunostained for intracellular expression of p27 and analyzed by FACS. The percentage of SIV p27-positive cells, above the immunoglobulin control, is shown for each subset. (C) Immature DCs (human) were infected with live (ALVAC) or heat-inactivated (H.I.) ALVAC or left untreated (medium). After 4 days the DCs were examined for CD25 expression by FACS. The percentages of CD25-positive large cells (compared to the isotype control) are shown in each panel. The CD25-positive subset is highlighted by an arrowhead.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated monoclonal Abs (MAbs) against human major histocompatibility complex (MHC) class II (anti-HLA-DR-FITC) (Becton Dickinson Immunocytometry Systems [BDIS], San Jose, Calif.) were used in combination with phycoerythrin (PE)-conjugated MAbs against human CD25 (BDIS), CD80 (BDIS), CD83 (Coulter Corp., Miami, Fla.), and CD86 (PharMingen, San Diego, Calif.).

Techniques: Infection, Expressing

Journal:

Article Title: Canarypox Virus-Induced Maturation of Dendritic Cells Is Mediated by Apoptotic Cell Death and Tumor Necrosis Factor Alpha Secretion

doi:

Figure Lengend Snippet: Inhibition of maturation of ALVAC-infected DCs by the addition of a caspase 3 inhibitor. A total of 250 or 500 μM Z-DEVD-FMK, or the equivalent dilution of DMSO diluent for the high dose (DMSO), were added to immature DCs 30 min before infection with ALVAC. Inhibition of maturation was assessed by the lack of CD25 expression 4 days after infection using the DMSO-treated cells as the 100% matured population. The data represent the mean percentages of CD25-expression (% CD25 pos.) of three experiments.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated monoclonal Abs (MAbs) against human major histocompatibility complex (MHC) class II (anti-HLA-DR-FITC) (Becton Dickinson Immunocytometry Systems [BDIS], San Jose, Calif.) were used in combination with phycoerythrin (PE)-conjugated MAbs against human CD25 (BDIS), CD80 (BDIS), CD83 (Coulter Corp., Miami, Fla.), and CD86 (PharMingen, San Diego, Calif.).

Techniques: Inhibition, Infection, Expressing

Journal:

Article Title: Canarypox Virus-Induced Maturation of Dendritic Cells Is Mediated by Apoptotic Cell Death and Tumor Necrosis Factor Alpha Secretion

doi:

Figure Lengend Snippet: Maturation of uninfected DCs by ALVAC-infected immature DCs. Immature DCs were infected with ALVAC, and free virus was washed out. Uninfected immature DCs that had been stained with the green fluorescent dye CMFDA were added to the infected (unstained) DCs at a ratio of 1:1 (Inf. DCs). As controls, the supernatant from infected cells (collected directly after washing off the virus) was added to CMFDA-stained cells (Sup't) or the green-uninfected cells were kept in medium (Medium). Maturation (highlighted by arrowheads) was assessed by CD25 expression 4 days later. The log PE is expressed on the y axes (CD25 versus the IgG control), and the CMFDA fluorescence intensity is expressed on the x axes. The results from one of two similar experiments are provided.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated monoclonal Abs (MAbs) against human major histocompatibility complex (MHC) class II (anti-HLA-DR-FITC) (Becton Dickinson Immunocytometry Systems [BDIS], San Jose, Calif.) were used in combination with phycoerythrin (PE)-conjugated MAbs against human CD25 (BDIS), CD80 (BDIS), CD83 (Coulter Corp., Miami, Fla.), and CD86 (PharMingen, San Diego, Calif.).

Techniques: Infection, Staining, Expressing, Fluorescence

Journal: Cell Reports Medicine

Article Title: Developing Human Skin Contains Lymphocytes Demonstrating a Memory Signature

doi: 10.1016/j.xcrm.2020.100132

Figure Lengend Snippet: Conventional αβ T Cells in Fetal Skin Largely Demonstrate a Naive, Proliferative Phenotype Live, CD4 + , and CD8 + single-positive cells from 23 weeks fetal skin and adult skin processed for CyTOF were identified by surface markers and analyzed as follows. (A–D) UMAP plots of combined fetal and adult (A) CD4 + and (B) CD8 + T cells colored by skin age. Principal component analysis (PCA) plots demonstrating the distribution of all (C) CD4 + and (D) CD8 + T cells from each individual sample by age. (E) UMAP plots of CD4 + T cells from fetal and adult skin, combined. Cells are labeled and colored by cluster, with clusters A and B constituting conventional CD4 + T cells and cluster C representing Tregs. (F and G) Analogous UMAP plots containing only (F) fetal or (G) adult CD4 + T cells. (H) Heatmap demonstrating relative expression of key markers by CD4 + clusters A, B, and C. (I–K) combined and separated UMAP plots of fetal and adult skin CD8 + T cells, colored by cluster. (L) Heatmap demonstrating relative expression of key markers by each CD8 + cluster. (M–R) Median expression intensity (m.e.i.) of Ki-67 (M and P), CD45RO (N and Q), and CD25 (O and R) for fetal versus adult CD4 + Tconv and CD8 + T cells as revealed by mass cytometric analyses. Each point in (C), (D), and (M)–(R) represents data from an individual donor. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Article Snippet: Anti-human CD25-PECy7 (clone: M-A251) , BD Biosciences , Cat# 557741; RRID: AB_396847.

Techniques: Labeling, Expressing

Journal: Cell Reports Medicine

Article Title: Developing Human Skin Contains Lymphocytes Demonstrating a Memory Signature

doi: 10.1016/j.xcrm.2020.100132

Figure Lengend Snippet: Subsets of Fetal Skin CD4 + Tconv and CD8 + T Cells Display a Memory Phenotype and Demonstrate Capacity for IFNγ Production Cells were isolated from second trimester fetal skin (scalp and/or torso) as well as adult (torso) skin and analyzed by flow cytometry. (A) Representative plots demonstrating CD45RO expression by fetal CD8 + T cells (gated on live CD3 + CD8 + CD4 neg ) and CD4 + Tconv (gated on live CD3 + CD4 + CD8 neg Foxp3 neg CD25 lo ). (B and C) Percentage of CD45RO + (B) CD8 + T cells and (C) CD4 + Tconv in fetal versus adult skin. (D–F) Representative histogram (D) and quantification (E and F) of Nur77 MFI on fetal skin CD45RO + versus CD45RA + CD4 + Tconv and CD8 + cells. (G–J) Percentage of CD4 + Tconv producing (G) IL-17A, (H) IL-13, and (I) IFNγ after PMA/ionomycin re-stimulation, and (J) percentage of IFNγ-producing CD8 + T cells. Each point in (B)–(G) represents data from an individual tissue sample; for some fetal samples data from scalp and torso skin from the same fetal donor are included as separate points. ns, not significant (p > 0.05); ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Article Snippet: Anti-human CD25-PECy7 (clone: M-A251) , BD Biosciences , Cat# 557741; RRID: AB_396847.

Techniques: Isolation, Flow Cytometry, Expressing

Journal: Cell Reports Medicine

Article Title: Developing Human Skin Contains Lymphocytes Demonstrating a Memory Signature

doi: 10.1016/j.xcrm.2020.100132

Figure Lengend Snippet: Tregs in Human Fetal Skin Demonstrate an Effector Memory Phenotype Twenty-three weeks g.a. fetal torso skin and healthy adult torso skin samples were analyzed for 22 markers using mass cytometry. (A) PCA plot of Tregs (cluster C, Figure 2 ) and CD4 + Tconv (clusters A & B, Figure 2 ) from 23 week fetal skin versus adult skin based on relative expression of key markers as assessed by mass cytometry. (B–E) Graphs showing median expression intensity (m.e.i.) of (B) Foxp3, (C) CD25, (D) PD-1, and (E) CTLA4 by mass cytometry on fetal versus adult skin Tregs. (F and G) Cells were isolated from 17–23 weeks g.a. fetal skin (scalp and/or torso) as well as adult (torso) skin and analyzed by flow cytometry. (F) Percentage of CD45RO + Tregs in skin by age and (G) accompanying representative flow plots. (H) Representative histogram and (i) quantification of Nur77 MFI on fetal skin CD45RO + versus CD45RA + Tregs. (J) Nur77 in fetal skin CD45RA + CD8 + , CD4 + Tconv, and Tregs. Each point in (A)–(E) represents data from an individual donor. Points in (F), (I), and (J) point represent data from an individual tissue sample; for some fetal samples data from scalp and torso skin from the same fetal donor are included as separate points. ns, not significant (p > 0.05); ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Article Snippet: Anti-human CD25-PECy7 (clone: M-A251) , BD Biosciences , Cat# 557741; RRID: AB_396847.

Techniques: Mass Cytometry, Expressing, Isolation, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Developing Human Skin Contains Lymphocytes Demonstrating a Memory Signature

doi: 10.1016/j.xcrm.2020.100132

Figure Lengend Snippet:

Article Snippet: Anti-human CD25-PECy7 (clone: M-A251) , BD Biosciences , Cat# 557741; RRID: AB_396847.

Techniques: Flow Cytometry, Mass Cytometry, Immunohistochemistry, Recombinant, Staining, Cell Stimulation, Software

Journal:

Article Title: The Generation of Monoclonal Antibodies Specific for Cell Surface Molecules Expressed on Early Mouse Endoderm

doi: 10.1002/stem.147

Figure Lengend Snippet: (A) Schematic of Foxa3 targeting vector. (B) Southern blot analyses identifying a targeted clone and a targeted clone following removal of the selection cassette (I delta puro). (C) Kinetic analyses of CD4-Foxa2 and CD25-Foxa3 expression during endoderm formation in EBs generated from the CD25-Foxa3 reporter cell line. (D) QPCR-based gene expression analysis of CD4+CD25+ and CD4+CD25− populations isolated from day six EBs. Extra-embryonic tissue from day 8.25 mouse embryos was used as a control. Relative expression is shown with the presort population set to 1 except for the extra-embryonic endoderm genes where EE was set to 1. (E) Expression of CD4-Foxa2 and CD25-Foxa3 on CD4+CD25− and CD4+CD25+-derived populations following 12 days of culture in hepatic inducing conditions. One of three independent experiments is shown (C-E). Abbreviations: DTA, diphtheria toxin-A gene; pA, bovine poly-adenylation sequence; Puro, puromycin resistance cassette; −Con, negative control; CD25−, CD4+CD25− population; CD25+, CD4+CD25+ population; EE, extra-embryonic tissue.

Article Snippet: Flow Cytometry and Cell Sorting Various ES cell derived cultures or mouse embryos were dissociated by incubation with trypsin for 1-3 minutes and stained for the following cell surface antigens: anti-human CD4-phycoerythrin, −allophycocyanin, or −phycoerythrin/Cy5.5 (Invitrogen), anti-human CD25-phycoerythrin or −allophycocyanin (Invitrogen), anti-mouse EpCAM-phycoerythrin (Santa Cruz Biotechnology), or anti-mouse CXCR4-biotin (Becton Dickenson) followed by streptavidin–phycoerythrin/Cy5.5 (Invitrogen).

Techniques: Plasmid Preparation, Southern Blot, Selection, Expressing, Generated, Isolation, Derivative Assay, Sequencing, Negative Control

Journal:

Article Title: The Generation of Monoclonal Antibodies Specific for Cell Surface Molecules Expressed on Early Mouse Endoderm

doi: 10.1002/stem.147

Figure Lengend Snippet: (A) Staining patterns of CD25-Foxa3, ENDM1 and ENDM2 on ES cell-derived endoderm, mesoderm, ectoderm and on undifferentiated ES cells. EBs differentiated in various conditions for 6 days were analyzed. (B) Staining patterns of CD4-Foxa2 versus CD25-Foxa3, ENDM1 and ENDM2 on sorted endoderm cells cultured in hepatocyte conditions for 1 day (day 6 total), 3 days (day 8 total) or 5 days (day 10 total). Histograms shown are gated on CD4-Foxa2+ CD25-Foxa3+ cells. (C) Afp and HNF6 expression (QPCR) in cells cultured as described in B. Relative expression is shown with the D10 population set to 1. Abbreviations: ES, ES cells; FL, day 15 fetal liver; D6, Day 6; D8, Day 8; D10, Day 10.

Article Snippet: Flow Cytometry and Cell Sorting Various ES cell derived cultures or mouse embryos were dissociated by incubation with trypsin for 1-3 minutes and stained for the following cell surface antigens: anti-human CD4-phycoerythrin, −allophycocyanin, or −phycoerythrin/Cy5.5 (Invitrogen), anti-human CD25-phycoerythrin or −allophycocyanin (Invitrogen), anti-mouse EpCAM-phycoerythrin (Santa Cruz Biotechnology), or anti-mouse CXCR4-biotin (Becton Dickenson) followed by streptavidin–phycoerythrin/Cy5.5 (Invitrogen).

Techniques: Staining, Derivative Assay, Cell Culture, Expressing

Journal:

Article Title: The Generation of Monoclonal Antibodies Specific for Cell Surface Molecules Expressed on Early Mouse Endoderm

doi: 10.1002/stem.147

Figure Lengend Snippet: (A) CD4-Foxa2 versus CD25-Foxa3 expression on CD4+CD25+, CD4+CD25−, ENDM1+, ENDM1−, ENDM2+, and ENDM2− derived cells following 10 days of culture in hepatocyte inducing conditions. Populations were isolated from day 5 EBs induced under endoderm conditions. (B) Intracellular flow cytometry measuring the proportion of CD4-Foxa2+ and albumin expressing cells in the CD4+CD25+, ENDM1+ and ENDM2+-derived populations shown in A. (C) Intracellular flow cytometry measuring the proportion of CD4-Foxa2+ and Afp expressing cells in the CD4+CD25+, ENDM1+ and ENDM2+-derived populations shown in A. (D) Afp and HNF6 expression in the different populations described in A by QPCR. (E) Expression of Foxa1 and HNF4a in ENDM1+, ENDM1−, ENDM2+, and ENDM2− populations sorted from 5-day-old EBs generated from E14 wild type ES cells differentiated in endoderm inducing conditions (QPCR). Relative expression is shown with the presort population set to 1. Abbreviations: CD4, CD4-Foxa2; 25, CD25-Foxa3; E1, ENDM1; E2, ENDM2; Pre, presort.

Article Snippet: Flow Cytometry and Cell Sorting Various ES cell derived cultures or mouse embryos were dissociated by incubation with trypsin for 1-3 minutes and stained for the following cell surface antigens: anti-human CD4-phycoerythrin, −allophycocyanin, or −phycoerythrin/Cy5.5 (Invitrogen), anti-human CD25-phycoerythrin or −allophycocyanin (Invitrogen), anti-mouse EpCAM-phycoerythrin (Santa Cruz Biotechnology), or anti-mouse CXCR4-biotin (Becton Dickenson) followed by streptavidin–phycoerythrin/Cy5.5 (Invitrogen).

Techniques: Expressing, Derivative Assay, Isolation, Flow Cytometry, Generated

Journal: iScience

Article Title: Defects in NK cell immunity of pediatric cancer patients revealed by deep immune profiling

doi: 10.1016/j.isci.2024.110837

Figure Lengend Snippet:

Article Snippet: Anti-CD25 (clone 2A3, conjugated to 149Sm) , Standard BioTools , Cat#3149010B.

Techniques: Purification, Clinical Proteomics, Recombinant, Blocking Assay, Staining, Saline, Mass Cytometry, Software, Cytometry