Journal:

Article Title: Suppression of HIV-specific T cell activity by lymph node CD25 + regulatory T cells from HIV-infected individuals

doi: 10.1073/pnas.0611423104

Figure Lengend Snippet: Representative phenotypic analysis of CD25 and FoxP3 expression in CD4+ T cells of PBMC compared with LNMC. (A) Freshly isolated PBMC and LNMC were stained for CD3, CD4, CD25 and intracellular FoxP3. CD25 and FoxP3 expression was analyzed in the CD3+CD4+ population. (A) CD25+ Treg cells in PBMC and LNMC were quantified as FoxP3+ and CD25hi (MFI ≥70) as indicated in the box in the upper right quadrant. (B) Representative example of CD25+hi expression on purified CD4+ T cell subsets used in functional assays. (C) Representative example of FoxP3 expression intensity (MFI) on CD25+ CD4+ T cells in LN and PB.

Article Snippet: CD25 hi cells were obtained by using anti-human CD25 PE conjugated Mab (3 μl/10 6 cells) followed by a short incubation (3 min) with anti-PE immunomagnetic beads (Miltenyi, Auburn, CA).

Techniques: Expressing, Isolation, Staining, Purification, Functional Assay

Journal:

Article Title: Suppression of HIV-specific T cell activity by lymph node CD25 + regulatory T cells from HIV-infected individuals

doi: 10.1073/pnas.0611423104

Figure Lengend Snippet: The frequency and MFI of FoxP3 expression in CD4+ T cell subsets within LNMC is significantly greater than within PBMC. CD25hi (Top) and CD25− (Middle) FoxP3+ CD4+ T cell frequencies and FoxP3 MFI in CD25+ CD4+ T cells (Bottom) in parallel LNMC and PBMC isolated from 11 chronically HIV-infected individuals.

Article Snippet: CD25 hi cells were obtained by using anti-human CD25 PE conjugated Mab (3 μl/10 6 cells) followed by a short incubation (3 min) with anti-PE immunomagnetic beads (Miltenyi, Auburn, CA).

Techniques: Expressing, Isolation, Infection

Journal:

Article Title: Suppression of HIV-specific T cell activity by lymph node CD25 + regulatory T cells from HIV-infected individuals

doi: 10.1073/pnas.0611423104

Figure Lengend Snippet: Comparison of PB and LN for the expression of surface markers used to distinguish CD25+ Treg cells and normal CD4+ T cells. LNMC and PBMC were stained for CD4, CD25, intracellular FoxP3, and either sCTLA-4, GITR, or CD127. CD25−FoxP3− and CD25hiFoxP3+ CD4+ LNMC and PBMC subsets were analyzed for expression of CD127, sCTLA-4, and GITR. P values compare LNMC and PBMC subsets. Data are of mean percent ± SD of data obtained from LNMC and PBMC isolated from 11 chronically HIV-infected individuals.

Article Snippet: CD25 hi cells were obtained by using anti-human CD25 PE conjugated Mab (3 μl/10 6 cells) followed by a short incubation (3 min) with anti-PE immunomagnetic beads (Miltenyi, Auburn, CA).

Techniques: Comparison, Expressing, Staining, Isolation, Infection

Journal:

Article Title: Suppression of HIV-specific T cell activity by lymph node CD25 + regulatory T cells from HIV-infected individuals

doi: 10.1073/pnas.0611423104

Figure Lengend Snippet: CD25+ Treg suppressive capacity assessed in HIV p24-specific proliferation assays. Total and CD25+ cell-depleted CD4+ T cells isolated from LNMC and PBMC of subjects 1–5 were stimulated with HIV p24 protein for 6 days, then pulsed with 3H thymidine 16 h. Percent suppression of proliferation by CD25+ cells was determined by comparing net cpm obtained in total versus CD25+ cell-depleted CD4+ T cell cultures (≥40% was arbitrarily considered to be a significant level of suppression). N.R., no response.

Article Snippet: CD25 hi cells were obtained by using anti-human CD25 PE conjugated Mab (3 μl/10 6 cells) followed by a short incubation (3 min) with anti-PE immunomagnetic beads (Miltenyi, Auburn, CA).

Techniques: Isolation

Journal:

Article Title: Suppression of HIV-specific T cell activity by lymph node CD25 + regulatory T cells from HIV-infected individuals

doi: 10.1073/pnas.0611423104

Figure Lengend Snippet: Representative example of CD25+ Treg suppressive capacity assessed in flow cytometry-based CTL assays by using Granzyme-B substrate cleavage as a read out. HIV Gag pre-stimulated effector unfractionated (Top plots) and CD25+ cell-depleted LNMC, alone or plus 30% CD25+CD8− MC, (Middle and Bottom Right plots, respectively) were cultured for 1 h with FL-4-labeled un-pulsed or HIV Gag peptide-pulsed autologous CD25−CD8− target cells in the presence of a Granzyme-B substrate that fluoresces when cleaved. Percent target cell killing (indicated within each plot) was defined by the frequency of substrate cleavage high events within the FL-4+ target cell gate; analyses are of FL-4+ target cells only. Substrate cleavage in target cells cultured in the absence of effector cells is shown in the Bottom Left plot.

Article Snippet: CD25 hi cells were obtained by using anti-human CD25 PE conjugated Mab (3 μl/10 6 cells) followed by a short incubation (3 min) with anti-PE immunomagnetic beads (Miltenyi, Auburn, CA).

Techniques: Flow Cytometry, Cell Culture, Labeling

Journal:

Article Title: Suppression of HIV-specific T cell activity by lymph node CD25 + regulatory T cells from HIV-infected individuals

doi: 10.1073/pnas.0611423104

Figure Lengend Snippet: Comparison of PB and LN CD25+ Treg cell-mediated suppression of HIV-specific CTL activity. (A) Mean (± SD) HIV-specific CTL activity in unfractionated and CD25+ cell-depleted PBMC and LNMC. (B) Mean (± SD) percent inhibition of HIV-specific CTL activity mediated by CD25+ cells in LNMC and PBMC of patients stratified by VL (HIV RNA copies per ml). (C) Comparison of the suppressive effects of LN and PB-derived CD25hi+ CD4+ T cells on HIV-specific CTL activity. Purified CD25− or CD25hi+ CD4+ T cells were isolated from the PB and LN (subjects 3–5) and added to autologous CD25+ cell-depleted MC (at 10%) at the time of primary stimulation with HIV Gag peptides. CTL assays were performed 6 days later after exposure to HIV Gag peptide-pulsed target cells. Data are of mean (±SD) percent suppression of HIV-specific CTL activity present in control CD25+ cell-depleted MC cultured alone.

Article Snippet: CD25 hi cells were obtained by using anti-human CD25 PE conjugated Mab (3 μl/10 6 cells) followed by a short incubation (3 min) with anti-PE immunomagnetic beads (Miltenyi, Auburn, CA).

Techniques: Comparison, Activity Assay, Inhibition, Derivative Assay, Purification, Isolation, Control, Cell Culture

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: The Journal of Experimental Medicine

Article Title: Expansion and tissue infiltration of an allospecific CD4 + CD25 + CD45RO + IL-7Rα high cell population in solid organ transplant recipients

doi: 10.1084/jem.20062120

Figure Lengend Snippet: Expression of CD25, CD45RO, IL-7Rα, and FOXP3 on blood CD4 T cells of healthy subjects and stable transplant recipients. Blood mononuclear cells from 45 healthy subjects and 32 stable organ transplant recipients (11 kidney and 21 liver transplant recipients) were analyzed for the surface expression of CD4, CD25, CD45RO, and IL-7Rα and for the intracellular expression of FOXP3. (A) Surface expression of CD45RO and IL-7Rα in CD4 + CD25 + T cells. Representative flow cytometry profiles of one healthy donor (1 out of 45; top) and one stable liver transplant recipient (1 out of 32; bottom). The vast majority of CD4 + CD25 + T cells are CD45RO + IL-7Rα low and FOXP3 + in the healthy donor, while a substantial percentage of CD4 + CD25 + T cells are CD45RO + IL-7Rα high and FOXP3 − (green) in the stable transplant recipient. (B) Cumulative data on the proportion of CD4 + CD25 + T cells (top left), CD4 + CD25 + FOXP3 + IL-7Rα low T cells (top right), and CD4 + CD25 + CD45RO + IL-7Rα high T cells (bottom) in stable transplant recipients and healthy donors. (C) Correlation between the expression of FOXP3 and IL-7Rα within CD4 + CD25 + T cells. The percentage of FOXP3 + cells correlated with that of IL-7Rα low cells within CD4 + CD25 + T cells, and the percentage of FOXP3 − cells correlated with that of IL-7Rα high cells within CD4 + CD25 + T cells. The analyses were performed in 26 stable transplant recipients. In both cases, these correlations were statistically significant (P < 0.05). (D) Suppressive activity of IL-7Rα high and IL-7Rα low CD4 + CD25 + CD45RO + T cell populations in stable transplant recipients. CD4 + CD25 + CD45RO + IL-7Rα high and CD4 + CD25 + CD45RO + IL-7Rα low cell populations were sorted from blood mononuclear cells of five stable liver transplant recipients, and their suppressive activity was evaluated in a MLR. The extent of cell proliferation in a MLR was assessed in the absence (positive control) or in presence of the sorted IL-7Rα low and IL-7Rα high cell populations. Cell proliferation was measured by [ 3 H]thymidine incorporation. The data shown are expressed as the mean ± SE and were obtained from five independent experiments.

Article Snippet: The antibodies used for flow cytometric analyses included PerCP, PerCP-Cy5.5, or PE-conjugated mouse anti–human CD4 (Becton Dickinson); allophycocyanin (APC)-conjugated mouse anti–human CD25 (BD Biosciences), FITC (BD Biosciences), or ECD (Beckman Coulter)-conjugated mouse anti–human CD45RO; and PE (Beckman Coulter)- or APC (R&D Systems)-conjugated mouse anti–human IL-7Rα.

Techniques: Expressing, Flow Cytometry, Activity Assay, Positive Control

Journal: The Journal of Experimental Medicine

Article Title: Expansion and tissue infiltration of an allospecific CD4 + CD25 + CD45RO + IL-7Rα high cell population in solid organ transplant recipients

doi: 10.1084/jem.20062120

Figure Lengend Snippet: Expression of CD25, CD45RO, IL-7Rα, and FOXP3 on blood CD4 T cells of stable transplant recipients and kidney transplant recipients with chronic rejection. Blood mononuclear cells from 32 stable transplant recipients (11 kidney and 21 liver transplant recipients) and 4 kidney transplant recipients with biopsy-proven chronic rejection were analyzed for the surface expression of CD4, CD25, CD45RO, and IL-7Rα and for the intracellular expression of FOXP3. (A) Means ± SE of cumulative data on the proportion of CD4 + CD25 + T cells in stable transplant recipients and transplant recipients with chronic rejection. The percentage of CD4 + CD25 + T cells in total CD4 + T cells was slightly increased in transplant recipients with chronic rejection compared with stable transplant recipients, but these differences were not significant (P = 0.21). (B) Means ± SE of cumulative data on the proportion of CD4 + CD25 + FOXP3 + IL-7Rα low T cells in stable transplant recipients and transplant recipients with chronic rejection. The proportion of FOXP3 + IL-7Rα low T cells within the CD4 + CD25 + T cell population was slightly reduced in the patients with chronic rejection compared with stable transplant recipients, and these differences were significant (P = 0.01).(C) Means ± SE of cumulative data on the proportion of CD4 + CD25 + CD45RO + IL-7Rα high T cells in stable transplant recipients and transplant recipients with chronic rejection. The proportion of CD45RO + IL-7Rα high T cells within the CD4 + CD25 + T cell population was significantly increased in the chronic rejection group compared with the stable group (P < 0.0001).

Article Snippet: The antibodies used for flow cytometric analyses included PerCP, PerCP-Cy5.5, or PE-conjugated mouse anti–human CD4 (Becton Dickinson); allophycocyanin (APC)-conjugated mouse anti–human CD25 (BD Biosciences), FITC (BD Biosciences), or ECD (Beckman Coulter)-conjugated mouse anti–human CD45RO; and PE (Beckman Coulter)- or APC (R&D Systems)-conjugated mouse anti–human IL-7Rα.

Techniques: Expressing

Journal: The Journal of Experimental Medicine

Article Title: Expansion and tissue infiltration of an allospecific CD4 + CD25 + CD45RO + IL-7Rα high cell population in solid organ transplant recipients

doi: 10.1084/jem.20062120

Figure Lengend Snippet: Analysis of allospecific CD4 T cell responses. Sorted purified CD4 + CD25 + CD45RO + IL-7Rα high T cells (7 × 10 4 cells) from two time points of a kidney transplant recipient with biopsy-proven chronic rejection were co-cultured with 10 5 irradiated blood mononuclear cells (40 Gy) either from the organ donor or from an MHC unrelated subject. After 7 d of culture, cell proliferation was measured by [ 3 H]thymidine incorporation. Cell cultures containing only irradiated cells were used as negative controls.

Article Snippet: The antibodies used for flow cytometric analyses included PerCP, PerCP-Cy5.5, or PE-conjugated mouse anti–human CD4 (Becton Dickinson); allophycocyanin (APC)-conjugated mouse anti–human CD25 (BD Biosciences), FITC (BD Biosciences), or ECD (Beckman Coulter)-conjugated mouse anti–human CD45RO; and PE (Beckman Coulter)- or APC (R&D Systems)-conjugated mouse anti–human IL-7Rα.

Techniques: Purification, Cell Culture, Irradiation

Journal: The Journal of Experimental Medicine

Article Title: Expansion and tissue infiltration of an allospecific CD4 + CD25 + CD45RO + IL-7Rα high cell population in solid organ transplant recipients

doi: 10.1084/jem.20062120

Figure Lengend Snippet: CD4 + IL-7Rα + T cells infiltrate the tissue allograft. Frozen kidney biopsies from transplant recipients with chronic rejection were stained with IL-7Rα and CD4 or CD25. (A, top) IL-7Rα + T cells (green) and CD4 + T cells (red) are indicated by the arrowheads and are present in the interstitial compartment. Double-positive CD4 + IL-7Rα + T cells (yellow arrowheads) are colored in orange as a result of the merging of the two single-color images. (middle) IL-7Rα + T cells (green) and CD25 + T cells (red) are also present in the interstitial compartment. Double-positive CD25 + IL-7Rα + T cells (yellow arrowheads) are colored in orange as a result of the merging of the two single-color images. (bottom) CD25 + T cells (green) and CD4 + T cells (red). Double-positive CD4 + CD25 + T cells (yellow arrowheads) are colored in orange as a result of the merging of the two single-color images. (B, top) CD4 + T cells (green) infiltrating the kidney of a patient with documented chronic rejection are FOXP3 negative (red). (middle and bottom) CD25 + T cells (green) and IL-7Rα + T cells (green) are also negative for FOXP3 (red). (C) CD4 + T cells (yellow) in the lymph node are FOXP3 positive (green). Bars, 10 μm.

Article Snippet: The antibodies used for flow cytometric analyses included PerCP, PerCP-Cy5.5, or PE-conjugated mouse anti–human CD4 (Becton Dickinson); allophycocyanin (APC)-conjugated mouse anti–human CD25 (BD Biosciences), FITC (BD Biosciences), or ECD (Beckman Coulter)-conjugated mouse anti–human CD45RO; and PE (Beckman Coulter)- or APC (R&D Systems)-conjugated mouse anti–human IL-7Rα.

Techniques: Staining

Journal: The Journal of Experimental Medicine

Article Title: Expansion and tissue infiltration of an allospecific CD4 + CD25 + CD45RO + IL-7Rα high cell population in solid organ transplant recipients

doi: 10.1084/jem.20062120

Figure Lengend Snippet: Rapid expansion of the CD4 + CD25 + CD45RO + IL-7Rα high cell population after transplantation. Means ± SE of cumulative data on the proportion of the CD4 + CD25 + CD45RO + IL-7Rα high T cell population in transplant recipients before and up to 1 yr after transplantation. At the baseline (before transplantation), recipients and donors have a similar proportion of CD4 + CD25 + CD45RO + IL-7Rα high T cells. The IL-7Rα high CD4 T cell population is already significantly increased 1 mo after transplantation in the transplant recipients and remains expanded for up to 1 yr.

Article Snippet: The antibodies used for flow cytometric analyses included PerCP, PerCP-Cy5.5, or PE-conjugated mouse anti–human CD4 (Becton Dickinson); allophycocyanin (APC)-conjugated mouse anti–human CD25 (BD Biosciences), FITC (BD Biosciences), or ECD (Beckman Coulter)-conjugated mouse anti–human CD45RO; and PE (Beckman Coulter)- or APC (R&D Systems)-conjugated mouse anti–human IL-7Rα.

Techniques: Transplantation Assay

Journal: PLoS ONE

Article Title: The Pronounced Th17 Profile in Systemic Sclerosis (SSc) Together with Intracellular Expression of TGFβ and IFNγ Distinguishes SSc Phenotypes

doi: 10.1371/journal.pone.0005903

Figure Lengend Snippet: Panel A presents the percentage of IL-23R positive cells in the CD4+ positive pool of T cells (left panel) and CD45Ro and CD45Ra positive cells (right panel) from healthy controls (n = 14), and SSc patients with the lcSSc (n = 12), ldcSSc (n = 11) and edcSSc (n = 13) phenotype. Panel B shows the mean intracellular expression level of IL-17, IFNγ and TGFβ in CD4+ cells from each a representative individual from each tested group (left side). On the right side, the mean percentage of CD45Ro-positive or CD45Ra-positive cells that express IL-17, IFNγ or TGFβ and the mean intensity thereof are presented for the whole group of healthy controls (n = 14), and SSc patients with the lcSSc (n = 12), ldcSSc (n = 11) and edcSSc (n = 13) phenotype. Panel C depicts the percentage of CD25 high cells that co-express IL-17 in healthy controls and different SSc phenotypes (* represents a p-value<0.01). Panel D represents the level of cytokines (IL-17 and IFNγ) spontaneously secreted by CD3+ T cells from healthy controls and SSc patients after 24 hrs of incubation. * P -value<0.0001, ** P -value<0.002, *** P -value<0.004.

Article Snippet: For immunostaining and analysis by fluorescence-activated cell sorting (FACS), we used phycoerythrin (PE), allophycocyanin (APC) and fluorescein isothiocynate (FITC) conjugated mouse monoclonal antibodies (mAb) against human CD4, CD8, GITR, CD69, IL-23R, CD45Ro, CD45Ra, and CD25 (Miltenyi Biotec Inc., CA, USA).

Techniques: Expressing, Incubation

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