cfse  (Thermo Fisher)


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    eBioscience CFSE
    Description:
    CFSE is widely used for cell tracking and proliferation studies It has also been used in CTL assays and cell motility studies CFSE readily crosses intact cell membranes Once inside the cells intracellular esterases cleave the acetate groups to yield the fluorescent carboxyfluorescein molecule The succinimidyl ester group reacts with primary amines crosslinking the dye to intracellular proteins Cell division can be measured as successive halving of the fluorescence intensity of CFSE Cells labeled with CFSE may be fixed and permeabilized for analysis of intracellular targets using standard formaldehyde containing fixatives and saponin based permeabilization buffers such as the Foxp3 Transcription Factor Staining Buffer Set cat 00 5523 or the IC Fixation Buffer cat 00 8222 and Permeabilization Buffer 10X cat 00 8333 CFSE has a molecular weight of 557 47 After the acetate groups are cleaved it has a peak excitation of 494 nm and peak emission of 521 nm Each vial of CFSE may be reconstituted to a stock concentration of 10 mM with 90 µL of anhydrous DMSO once reconstituted it should be used within 6 months and protected from light and stored at 20°C with dessicant avoid freeze thawing Reported ApplicationFlow Cytometric Analysis Microscopy
    Catalog Number:
    65-0850-84
    Price:
    None
    Category:
    Labeling Detection Products
    Applications:
    Cell Analysis|Flow Cytometry|Cell Viability, Proliferation & Function
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    Structured Review

    Thermo Fisher cfse
    FcγRIIa co‐stimulation selectively suppresses type I and III IFNs. DCs were stimulated with Poly I:C, either or not combined with c‐IgG (A–E), and co‐cultured with allogeneic naïve <t>CD8</t> + T cells (C–E). mRNA expression (at indicated time points) was determined by quantitative RT‐PCR. (A and B) Data shown are from one experiment, representative of three independent experiments. (C) Intracellular expression of granzyme B, IFN‐γ, or TNF was determined by flow cytometry. CD8 + T cell proliferation was determined by EdU incorporation (D) or <t>CFSE</t> labeling (E). (C–E) Data are pooled from three independent experiments, mean ± SEM. * p
    CFSE is widely used for cell tracking and proliferation studies It has also been used in CTL assays and cell motility studies CFSE readily crosses intact cell membranes Once inside the cells intracellular esterases cleave the acetate groups to yield the fluorescent carboxyfluorescein molecule The succinimidyl ester group reacts with primary amines crosslinking the dye to intracellular proteins Cell division can be measured as successive halving of the fluorescence intensity of CFSE Cells labeled with CFSE may be fixed and permeabilized for analysis of intracellular targets using standard formaldehyde containing fixatives and saponin based permeabilization buffers such as the Foxp3 Transcription Factor Staining Buffer Set cat 00 5523 or the IC Fixation Buffer cat 00 8222 and Permeabilization Buffer 10X cat 00 8333 CFSE has a molecular weight of 557 47 After the acetate groups are cleaved it has a peak excitation of 494 nm and peak emission of 521 nm Each vial of CFSE may be reconstituted to a stock concentration of 10 mM with 90 µL of anhydrous DMSO once reconstituted it should be used within 6 months and protected from light and stored at 20°C with dessicant avoid freeze thawing Reported ApplicationFlow Cytometric Analysis Microscopy
    https://www.bioz.com/result/cfse/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
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    cfse - by Bioz Stars, 2021-05
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    Images

    1) Product Images from "Fc gamma receptor IIa suppresses type I and III interferon production by human myeloid immune cells"

    Article Title: Fc gamma receptor IIa suppresses type I and III interferon production by human myeloid immune cells

    Journal: European Journal of Immunology

    doi: 10.1002/eji.201847615

    FcγRIIa co‐stimulation selectively suppresses type I and III IFNs. DCs were stimulated with Poly I:C, either or not combined with c‐IgG (A–E), and co‐cultured with allogeneic naïve CD8 + T cells (C–E). mRNA expression (at indicated time points) was determined by quantitative RT‐PCR. (A and B) Data shown are from one experiment, representative of three independent experiments. (C) Intracellular expression of granzyme B, IFN‐γ, or TNF was determined by flow cytometry. CD8 + T cell proliferation was determined by EdU incorporation (D) or CFSE labeling (E). (C–E) Data are pooled from three independent experiments, mean ± SEM. * p
    Figure Legend Snippet: FcγRIIa co‐stimulation selectively suppresses type I and III IFNs. DCs were stimulated with Poly I:C, either or not combined with c‐IgG (A–E), and co‐cultured with allogeneic naïve CD8 + T cells (C–E). mRNA expression (at indicated time points) was determined by quantitative RT‐PCR. (A and B) Data shown are from one experiment, representative of three independent experiments. (C) Intracellular expression of granzyme B, IFN‐γ, or TNF was determined by flow cytometry. CD8 + T cell proliferation was determined by EdU incorporation (D) or CFSE labeling (E). (C–E) Data are pooled from three independent experiments, mean ± SEM. * p

    Techniques Used: Cell Culture, Expressing, Quantitative RT-PCR, Flow Cytometry, Cytometry, Labeling

    2) Product Images from "A novel chimeric antigen receptor containing a JAK-STAT signaling domain mediates superior antitumor effects"

    Article Title: A novel chimeric antigen receptor containing a JAK-STAT signaling domain mediates superior antitumor effects

    Journal: Nature medicine

    doi: 10.1038/nm.4478

    The 28-ΔIL2RB-z (YXXQ) CAR-T cells show a superior proliferative capacity and maintain less differentiated memory T cell phenotypes following the antigen stimulation (a) The fold expansion of the CD4 + and CD8 + CAR-T cells was calculated 7 days after stimulation with NALM-6 or K562 (n=8; repeated measures one-way ANOVA with Tukey’s multiple comparisons test for the NALM-6 data, F=20.12 for CD4 + T cells, F=36.57 for CD8 + T cells, degree of freedom=39; paired t -test for comparison between the NALM-6 and K562 data in individual CAR-T cells, t=10.34 (CD4 + -28-z), t=8.16 (CD4+-BB-z), t=13.98 (CD4 + -28-ΔIL2RB-z (YXXQ)), t=10.26 (CD4 + -28-ΔIL2RB (FLSL)-z (YXXQ)), t=6.33 (CD4 + -28-ΔIL2RB-z), t=7.47 (CD8 + -28-z), t=10.57 (CD8 + -BB-z), t=9.88 (CD8 + -28-ΔIL2RB-z (YXXQ)), t=12.63 (CD8 + -28-ΔIL2RB (FLSL)-z (YXXQ)), t=7.44 (CD8 + -28-ΔIL2RB-z), degree of freedom=7). (b) CAR-T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with NALM-6. The mean fluorescence intensity of CFSE was analyzed three days following the stimulation (n=4; repeated measures one-way ANOVA with Tukey’s multiple comparisons test; F=36.84 for CD4 + T cells, F=38.44 for CD8 + T cells; degree of freedom=19). (c) The frequency of dead cells within CD4 + or CD8 + CAR-T cell population was analyzed by flow cytometry 24 hours after stimulation with NALM-6 (n=4; repeated measures one-way ANOVA with Tukey’s multiple comparisons test; F=53.83 for CD4 + T cells, F=114.1 for CD8 + T cells; degree of freedom=24). (d) The frequency of CD45RA + CD62L + CCR7 + cells in the CD8 + CAR-T cell population 7 days after stimulation with NALM-6 (n=9; repeated measures one-way ANOVA with Tukey’s multiple comparisons test; F=14.35; degree of freedom=44). (e) CAR-transduced T cells were stimulated with NALM-6 and cultured with or without 25 μM of S3I-201 (STAT3 inhibitor) and/or 5 μM of pimozide (STAT5 inhibitor) for 3 days. The fold expansion of the CD8 + CAR-T cells was analyzed on day 7 (n=6, repeated measures one-way ANOVA with Tukey’s multiple comparisons test for each condition; F=46.52 for DMSO, F=15.67 for S3I-201, F=19.17 for pimozide, F=1.85 for S3I-201+pimozide; degree of freedom=17). In a – e , data were collected from different donor samples. Horizontal lines indicate mean values. (f–h) NOD-scid IL2rγ null (NSG) mice were intravenously infused with NALM-6 and subsequently transplanted with CAR-T cells. The spleen cells isolated from the mice were restimulated with NALM-6 (●) or K562 (x), and the fold expansion after 7 days of culture (g) and cytokine production (h) of CD8 + CAR-T cells were analyzed. The data shown are the sum of two independent experiments (n=6 mice for each group, ordinary one-way ANOVA with Tukey’s multiple comparisons test; F=10.85 for fold expansion, F=12.42 for IL-2, F=2.17 for IFN-γ, F=0.81 for TNF-α, F=7.51 for IL-2 + IFN-γ + TNF-α + cells, degree of freedom=29). In g and h , horizontal lines show mean values ± s.d. ns, not significant.
    Figure Legend Snippet: The 28-ΔIL2RB-z (YXXQ) CAR-T cells show a superior proliferative capacity and maintain less differentiated memory T cell phenotypes following the antigen stimulation (a) The fold expansion of the CD4 + and CD8 + CAR-T cells was calculated 7 days after stimulation with NALM-6 or K562 (n=8; repeated measures one-way ANOVA with Tukey’s multiple comparisons test for the NALM-6 data, F=20.12 for CD4 + T cells, F=36.57 for CD8 + T cells, degree of freedom=39; paired t -test for comparison between the NALM-6 and K562 data in individual CAR-T cells, t=10.34 (CD4 + -28-z), t=8.16 (CD4+-BB-z), t=13.98 (CD4 + -28-ΔIL2RB-z (YXXQ)), t=10.26 (CD4 + -28-ΔIL2RB (FLSL)-z (YXXQ)), t=6.33 (CD4 + -28-ΔIL2RB-z), t=7.47 (CD8 + -28-z), t=10.57 (CD8 + -BB-z), t=9.88 (CD8 + -28-ΔIL2RB-z (YXXQ)), t=12.63 (CD8 + -28-ΔIL2RB (FLSL)-z (YXXQ)), t=7.44 (CD8 + -28-ΔIL2RB-z), degree of freedom=7). (b) CAR-T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with NALM-6. The mean fluorescence intensity of CFSE was analyzed three days following the stimulation (n=4; repeated measures one-way ANOVA with Tukey’s multiple comparisons test; F=36.84 for CD4 + T cells, F=38.44 for CD8 + T cells; degree of freedom=19). (c) The frequency of dead cells within CD4 + or CD8 + CAR-T cell population was analyzed by flow cytometry 24 hours after stimulation with NALM-6 (n=4; repeated measures one-way ANOVA with Tukey’s multiple comparisons test; F=53.83 for CD4 + T cells, F=114.1 for CD8 + T cells; degree of freedom=24). (d) The frequency of CD45RA + CD62L + CCR7 + cells in the CD8 + CAR-T cell population 7 days after stimulation with NALM-6 (n=9; repeated measures one-way ANOVA with Tukey’s multiple comparisons test; F=14.35; degree of freedom=44). (e) CAR-transduced T cells were stimulated with NALM-6 and cultured with or without 25 μM of S3I-201 (STAT3 inhibitor) and/or 5 μM of pimozide (STAT5 inhibitor) for 3 days. The fold expansion of the CD8 + CAR-T cells was analyzed on day 7 (n=6, repeated measures one-way ANOVA with Tukey’s multiple comparisons test for each condition; F=46.52 for DMSO, F=15.67 for S3I-201, F=19.17 for pimozide, F=1.85 for S3I-201+pimozide; degree of freedom=17). In a – e , data were collected from different donor samples. Horizontal lines indicate mean values. (f–h) NOD-scid IL2rγ null (NSG) mice were intravenously infused with NALM-6 and subsequently transplanted with CAR-T cells. The spleen cells isolated from the mice were restimulated with NALM-6 (●) or K562 (x), and the fold expansion after 7 days of culture (g) and cytokine production (h) of CD8 + CAR-T cells were analyzed. The data shown are the sum of two independent experiments (n=6 mice for each group, ordinary one-way ANOVA with Tukey’s multiple comparisons test; F=10.85 for fold expansion, F=12.42 for IL-2, F=2.17 for IFN-γ, F=0.81 for TNF-α, F=7.51 for IL-2 + IFN-γ + TNF-α + cells, degree of freedom=29). In g and h , horizontal lines show mean values ± s.d. ns, not significant.

    Techniques Used: Labeling, Fluorescence, Flow Cytometry, Cytometry, Cell Culture, Mouse Assay, Isolation

    3) Product Images from "HIV-1 Nef Enhances Dendritic Cell-Mediated Viral Transmission to CD4+ T Cells and Promotes T-Cell Activation"

    Article Title: HIV-1 Nef Enhances Dendritic Cell-Mediated Viral Transmission to CD4+ T Cells and Promotes T-Cell Activation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0034521

    HIV-1-infected DCs cannot significantly increase resting CD4 + T cell proliferation in DC-T cell co-cultures. (A) Schematic representation of the experiment procedures. Primary MDDCs and autologous resting CD4 + T cells were used. (B) DCs infected with replication-competent WT or Nef-mutated HIV-1 were co-cultured with uninfected resting CD4 + T cells and HIV-1 p24 production in the supernatants was measured at the indicated times of co-cultures. Immature DCs were generated from purified monocytes by treatment with GM-CSF and IL-4 for 5 days. Resting CD4 + T cells were isolated from PBMCs using immunomagnetic particles and were cultured in the presence of IL-2 for 8 days. (C) Co-culture with HIV-1-infected DCs increases the surface expression of the transient T-cell activation marker CD69 on resting CD4 + T cells. CD69 expression was assessed at 3 days of co-cultures by flow cytometry. Mock-infected cells were used as background controls. These data are donor matched to Figure 6B . (D) CD4 + T cell proliferation in the co-cultures of HIV-1 infected DCs. DCs were mock infected or infected with WT or Nef-mutated HIV-1 for 3 days prior to co-culture with CFSE-labeled, uninfected resting CD4 + T cells. T cell proliferation was measured by flow cytometry at the indicated times of co-cultures. Data represent five independent experiments using cells from five different donors. Each symbol in the plots represents a single experiment result and the horizontal bars in the plots indicate the mean values of five independent experiments.
    Figure Legend Snippet: HIV-1-infected DCs cannot significantly increase resting CD4 + T cell proliferation in DC-T cell co-cultures. (A) Schematic representation of the experiment procedures. Primary MDDCs and autologous resting CD4 + T cells were used. (B) DCs infected with replication-competent WT or Nef-mutated HIV-1 were co-cultured with uninfected resting CD4 + T cells and HIV-1 p24 production in the supernatants was measured at the indicated times of co-cultures. Immature DCs were generated from purified monocytes by treatment with GM-CSF and IL-4 for 5 days. Resting CD4 + T cells were isolated from PBMCs using immunomagnetic particles and were cultured in the presence of IL-2 for 8 days. (C) Co-culture with HIV-1-infected DCs increases the surface expression of the transient T-cell activation marker CD69 on resting CD4 + T cells. CD69 expression was assessed at 3 days of co-cultures by flow cytometry. Mock-infected cells were used as background controls. These data are donor matched to Figure 6B . (D) CD4 + T cell proliferation in the co-cultures of HIV-1 infected DCs. DCs were mock infected or infected with WT or Nef-mutated HIV-1 for 3 days prior to co-culture with CFSE-labeled, uninfected resting CD4 + T cells. T cell proliferation was measured by flow cytometry at the indicated times of co-cultures. Data represent five independent experiments using cells from five different donors. Each symbol in the plots represents a single experiment result and the horizontal bars in the plots indicate the mean values of five independent experiments.

    Techniques Used: Infection, Cell Culture, Generated, Purification, Isolation, Co-Culture Assay, Expressing, Activation Assay, Marker, Flow Cytometry, Cytometry, Labeling

    Nef promotion of HIV-1 replication in primary resting CD4 + T cells is associated with T cell proliferation. Resting CD4 + T cells were isolated from PBMCs using immunomagnetic particles and were cultured in the presence of IL-2 before and during HIV-1 infection. (A) Resting CD4 + T cells were cultured for 8 days and then infected with 5 ng p24 of replication-competent WT or Nef-mutated HIV-1. HIV-1 p24 production in the supernatants was measured at the times indicated. Representative data from one donor are shown. (B) Resting CD4 + T cells were cultured for 8 days and then infected with 5 ng p24 of WT HIV-1 or Nef-mutated viruses. HIV-1 p24 production in the supernatant was measured at the times indicated. Mean values of two independent experiments on different donors are shown. (C) CFSE-labeled resting CD4 + T cells (cultured for 8 days in the presence of IL-2) were mock infected or infected with 5 ng p24 of WT or Nef-mutated HIV-1. CD4 + T cell proliferation was measured by flow cytometry at the indicated times. Data represent three independent experiments using cells from three different donors. Each symbol in the plots represents a single experiment result and the horizontal bars in the plots indicate the mean values of three independent experiments. There is no statistically significant difference in T-cell proliferation among WT and Nef-mutated HIV-1 infected CD4 + T cells.
    Figure Legend Snippet: Nef promotion of HIV-1 replication in primary resting CD4 + T cells is associated with T cell proliferation. Resting CD4 + T cells were isolated from PBMCs using immunomagnetic particles and were cultured in the presence of IL-2 before and during HIV-1 infection. (A) Resting CD4 + T cells were cultured for 8 days and then infected with 5 ng p24 of replication-competent WT or Nef-mutated HIV-1. HIV-1 p24 production in the supernatants was measured at the times indicated. Representative data from one donor are shown. (B) Resting CD4 + T cells were cultured for 8 days and then infected with 5 ng p24 of WT HIV-1 or Nef-mutated viruses. HIV-1 p24 production in the supernatant was measured at the times indicated. Mean values of two independent experiments on different donors are shown. (C) CFSE-labeled resting CD4 + T cells (cultured for 8 days in the presence of IL-2) were mock infected or infected with 5 ng p24 of WT or Nef-mutated HIV-1. CD4 + T cell proliferation was measured by flow cytometry at the indicated times. Data represent three independent experiments using cells from three different donors. Each symbol in the plots represents a single experiment result and the horizontal bars in the plots indicate the mean values of three independent experiments. There is no statistically significant difference in T-cell proliferation among WT and Nef-mutated HIV-1 infected CD4 + T cells.

    Techniques Used: Isolation, Cell Culture, Infection, Labeling, Flow Cytometry, Cytometry

    4) Product Images from "Sequential transcriptional changes dictate safe and effective antigen-specific immunotherapy"

    Article Title: Sequential transcriptional changes dictate safe and effective antigen-specific immunotherapy

    Journal: Nature Communications

    doi: 10.1038/ncomms5741

    Dose escalation to higher peptide doses improves induction of a regulatory CD4 + T-cell phenotype. ( a ) Treatment groups for EDI with increasingly higher final doses. ( b ) Proliferative responses of CD4 + T cells from EDI-treated Tg4 mice cultured with irradiated antigen-presenting cells (APCs) and MBP Ac1-9[4K] for 3 days, measured by 3 [H] thymidine incorporation. Representative of three independent experiments each with two biological replicates assayed in triplicate. ( c ) Percentages of Vβ8 + T cells expressing IL-10 and IFN-γ after 6 days culture with 10 μg ml −1 MBP Ac1-9[4K], detected by flow cytometric analysis. Representative of two similar experiments, error bars show+s.e.m. of two biological replicates. ( d ) For in vitro suppression assay, in vitro -expanded CD4 + T cells from peptide-treated Tg4 mice cultured 1:1 with carboxyfluorescein succinimidyl ester (CFSE)-labelled responder CD4 + T cells, APCs and 10 μg ml −1 MBP Ac1-9[4K]. After 3 days, the proliferative response of CD4 + CFSE + responder cells was measured by flow cytometry. For in vivo suppression assay, 5 × 10 6 Cell Trace Violet (CTV)-labelled CD45.1 + Tg4 CD4 + T cells were transferred i.v. into EDI-treated Tg4 CD45.2 + mice. After 24 h, mice were injected s.c. with 80 μg of MBP Ac1-9[4Y]. Three days after peptide challenge, Cell Trace Violet-labelled CD45.1 + CD4 + cells were recovered from spleens for flow cytometric analysis. Data in each plot are representative of two biological replicates, offset histograms show proliferation dye dilution and division indexes.
    Figure Legend Snippet: Dose escalation to higher peptide doses improves induction of a regulatory CD4 + T-cell phenotype. ( a ) Treatment groups for EDI with increasingly higher final doses. ( b ) Proliferative responses of CD4 + T cells from EDI-treated Tg4 mice cultured with irradiated antigen-presenting cells (APCs) and MBP Ac1-9[4K] for 3 days, measured by 3 [H] thymidine incorporation. Representative of three independent experiments each with two biological replicates assayed in triplicate. ( c ) Percentages of Vβ8 + T cells expressing IL-10 and IFN-γ after 6 days culture with 10 μg ml −1 MBP Ac1-9[4K], detected by flow cytometric analysis. Representative of two similar experiments, error bars show+s.e.m. of two biological replicates. ( d ) For in vitro suppression assay, in vitro -expanded CD4 + T cells from peptide-treated Tg4 mice cultured 1:1 with carboxyfluorescein succinimidyl ester (CFSE)-labelled responder CD4 + T cells, APCs and 10 μg ml −1 MBP Ac1-9[4K]. After 3 days, the proliferative response of CD4 + CFSE + responder cells was measured by flow cytometry. For in vivo suppression assay, 5 × 10 6 Cell Trace Violet (CTV)-labelled CD45.1 + Tg4 CD4 + T cells were transferred i.v. into EDI-treated Tg4 CD45.2 + mice. After 24 h, mice were injected s.c. with 80 μg of MBP Ac1-9[4Y]. Three days after peptide challenge, Cell Trace Violet-labelled CD45.1 + CD4 + cells were recovered from spleens for flow cytometric analysis. Data in each plot are representative of two biological replicates, offset histograms show proliferation dye dilution and division indexes.

    Techniques Used: Mouse Assay, Cell Culture, Irradiation, Expressing, Flow Cytometry, In Vitro, Suppression Assay, Cytometry, In Vivo, Injection

    5) Product Images from "SLAP Deficiency Enhances Number and Function of Regulatory T Cells Preventing Chronic Autoimmune Arthritis in SKG Mice"

    Article Title: SLAP Deficiency Enhances Number and Function of Regulatory T Cells Preventing Chronic Autoimmune Arthritis in SKG Mice

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

    doi: 10.4049/jimmunol.1003601

    Functional blockade of Tregs in DSSKO mice leads to the development of arthritis. A , Arthritis development in mice injected i.v. with a mixture of anti-CD25 Abs or control rat Ig (black arrows) on days 5, 7, and 9 after zymosan treatment. Data represent the average arthritis score (±SEM) from three independent experiments for SKG and DSSKO mice. Data for WT and SLAP knockout mice are from one experiment. B and C, CFSE-labeled WT CD4 + CD25 − peripheral T cells were stimulated with anti-CD3 and anti-CD28 in the presence of the indicated ratios of either freshly isolated ( B ) or activated ( C ) CD4 + CD25 + Tregs from WT, SLAP −/− , SKG, or DSSKO mice and proliferation determined by dilution of CFSE using flow cytometry. Data represent the average of three mice per genotype (±SEM) from three independent experiments.
    Figure Legend Snippet: Functional blockade of Tregs in DSSKO mice leads to the development of arthritis. A , Arthritis development in mice injected i.v. with a mixture of anti-CD25 Abs or control rat Ig (black arrows) on days 5, 7, and 9 after zymosan treatment. Data represent the average arthritis score (±SEM) from three independent experiments for SKG and DSSKO mice. Data for WT and SLAP knockout mice are from one experiment. B and C, CFSE-labeled WT CD4 + CD25 − peripheral T cells were stimulated with anti-CD3 and anti-CD28 in the presence of the indicated ratios of either freshly isolated ( B ) or activated ( C ) CD4 + CD25 + Tregs from WT, SLAP −/− , SKG, or DSSKO mice and proliferation determined by dilution of CFSE using flow cytometry. Data represent the average of three mice per genotype (±SEM) from three independent experiments.

    Techniques Used: Functional Assay, Mouse Assay, Injection, Knock-Out, Labeling, Isolation, Flow Cytometry, Cytometry

    6) Product Images from "Healthy individuals have T-cell and antibody responses to the tumor antigen cyclin B1 that when elicited in mice protect from cancer"

    Article Title: Healthy individuals have T-cell and antibody responses to the tumor antigen cyclin B1 that when elicited in mice protect from cancer

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0903225106

    Healthy individuals have memory T cells specific for cyclin B1. ( A ) All T cells: Monocyte-depleted PBMC were cultured with autologous DCs that were loaded with ovalbumin (OVA), cyclin B1 (CB1), or unloaded. Supernatant from the seventh day of culture was tested by ELISA for IFNγ. W6/32: MHC class I blocking antibody. ( B ) CD4 + T cells: PBMC from the same donor as in A were labeled with CFSE and cultured with autologous DCs in the presence or absence of indicated antigen or without DC. Percentage of proliferating CD4 + T cells was assessed after seven days. ( C ) CD8 + T cells: CD8 + T cells were purified from PBMC (a second donor) and cultured with autologous DCs with and without indicated antigens. Supernatants were tested for IFNy after 10 days. Bars indicate standard deviation. ( D–G ) Brefeldin A was added for 11 h to one set of a triplicate culture at 6, 30, and 54 h after combination of DCs with PBL. After the incubation periods, CD4 + T cells ( D and F ) and CD8 + T cells ( E and G ) were assessed for intracellular IFNγ. ( D and E ) Flow cytometric measurement of IFNγ. ( Top ) PBL stimulated with cyclin B1-loaded DCs. ( Center ) PBL stimulated with OVA-loaded DCs. ( Lower ) PBL stimulated with unloaded DCs. ( F and G ) show a graphical representation of the percentage of IFNγ-positive cells for CD4 + ( F ) and CD8 + ( G ) T cells.
    Figure Legend Snippet: Healthy individuals have memory T cells specific for cyclin B1. ( A ) All T cells: Monocyte-depleted PBMC were cultured with autologous DCs that were loaded with ovalbumin (OVA), cyclin B1 (CB1), or unloaded. Supernatant from the seventh day of culture was tested by ELISA for IFNγ. W6/32: MHC class I blocking antibody. ( B ) CD4 + T cells: PBMC from the same donor as in A were labeled with CFSE and cultured with autologous DCs in the presence or absence of indicated antigen or without DC. Percentage of proliferating CD4 + T cells was assessed after seven days. ( C ) CD8 + T cells: CD8 + T cells were purified from PBMC (a second donor) and cultured with autologous DCs with and without indicated antigens. Supernatants were tested for IFNy after 10 days. Bars indicate standard deviation. ( D–G ) Brefeldin A was added for 11 h to one set of a triplicate culture at 6, 30, and 54 h after combination of DCs with PBL. After the incubation periods, CD4 + T cells ( D and F ) and CD8 + T cells ( E and G ) were assessed for intracellular IFNγ. ( D and E ) Flow cytometric measurement of IFNγ. ( Top ) PBL stimulated with cyclin B1-loaded DCs. ( Center ) PBL stimulated with OVA-loaded DCs. ( Lower ) PBL stimulated with unloaded DCs. ( F and G ) show a graphical representation of the percentage of IFNγ-positive cells for CD4 + ( F ) and CD8 + ( G ) T cells.

    Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay, Blocking Assay, Labeling, Purification, Standard Deviation, Incubation, Flow Cytometry

    7) Product Images from "Cross-presenting CD103+ dendritic cells are protected from influenza virus infection"

    Article Title: Cross-presenting CD103+ dendritic cells are protected from influenza virus infection

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI60659

    Lung tissue migratory CD103 + DCs control anti-viral CD8 + T cell immunity. ( A ) WT and Ccr7 –/– mice were infected with 10 7 PFUs of PR8-OTI virus. Forty-eight hours later, MLN cells were cocultured with CFSE-labeled CD8 + OT-I T cells for 3 days. Proliferation was measured by flow cytometry assessment of CFSE dilution (representative histograms). Graph represents the number of divided T cells per well. Each dot represents 1 mouse. ( B ) WT mice were infected with 10 7 PFUs of PR8-OTI virus. Lung migratory CD103 + DCs and CD11b + DCs and LN-resident CD8 + and CD4 + DCs purified from the MLN 24 hours after infection were cocultured with CFSE-labeled CD8 + OT-I T cells for 3 days. Proliferation was measured by flow cytometry assessment of CFSE dilution (representative histograms). Graph represents the number of divided T cells per well after coculture with different numbers of DCs per well (DC:T ratio). Data are representative of 3 separate experiments. ( C ) CD45 + CD11c + MHCII + lung phagocyte populations (CD103 + DCs, CD11b + DCs, and CD103 – CD11b – alveolar macrophages) were analyzed by flow cytometry in naive WT and Batf3 –/– mice. Representative percentages of each population in the CD45 + CD11c + MHCII + gate are noted. ( D and E ) WT and Batf3 –/– mice were infected with 3 × 10 3 PFUs of NS1-GFP virus. Seven days later, the percentages and absolute numbers of NP-specific endogenous CD8 + T cells in the MLNs ( D ) and the lungs ( E ) were measured by flow cytometry using H2D b /ASNENMETM-specific dextramer staining. Graphs represent the percentage (%) and the absolute numbers (abs no.) of dextramer-positive CD8 + T cells among total CD3 + CD8 + B220 – T cells. Data are representative of 3 separate experiments.
    Figure Legend Snippet: Lung tissue migratory CD103 + DCs control anti-viral CD8 + T cell immunity. ( A ) WT and Ccr7 –/– mice were infected with 10 7 PFUs of PR8-OTI virus. Forty-eight hours later, MLN cells were cocultured with CFSE-labeled CD8 + OT-I T cells for 3 days. Proliferation was measured by flow cytometry assessment of CFSE dilution (representative histograms). Graph represents the number of divided T cells per well. Each dot represents 1 mouse. ( B ) WT mice were infected with 10 7 PFUs of PR8-OTI virus. Lung migratory CD103 + DCs and CD11b + DCs and LN-resident CD8 + and CD4 + DCs purified from the MLN 24 hours after infection were cocultured with CFSE-labeled CD8 + OT-I T cells for 3 days. Proliferation was measured by flow cytometry assessment of CFSE dilution (representative histograms). Graph represents the number of divided T cells per well after coculture with different numbers of DCs per well (DC:T ratio). Data are representative of 3 separate experiments. ( C ) CD45 + CD11c + MHCII + lung phagocyte populations (CD103 + DCs, CD11b + DCs, and CD103 – CD11b – alveolar macrophages) were analyzed by flow cytometry in naive WT and Batf3 –/– mice. Representative percentages of each population in the CD45 + CD11c + MHCII + gate are noted. ( D and E ) WT and Batf3 –/– mice were infected with 3 × 10 3 PFUs of NS1-GFP virus. Seven days later, the percentages and absolute numbers of NP-specific endogenous CD8 + T cells in the MLNs ( D ) and the lungs ( E ) were measured by flow cytometry using H2D b /ASNENMETM-specific dextramer staining. Graphs represent the percentage (%) and the absolute numbers (abs no.) of dextramer-positive CD8 + T cells among total CD3 + CD8 + B220 – T cells. Data are representative of 3 separate experiments.

    Techniques Used: Mouse Assay, Infection, Labeling, Flow Cytometry, Cytometry, Purification, Staining

    Related Articles

    Cell Culture:

    Article Title: Epitope Hierarchy of Spontaneous CD4+ T Cell Responses to LAGE-1
    Article Snippet: Naive CD4+ and CD8+ T cells were purified from human PBMCs obtained from the blood of healthy donors by magnetic cell separation using CD45RA microbeads following the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA) and stained with CFSE (Invitrogen). .. CFSE-labeled naive T cells (1 × 105 ) were cultured for 5 d in 96-well flat bottom plates containing 2 × 105 irradiated (5000 rad) autologous CD3-depleted APCs, 0.1 µg/ml anti-CD3 (clone OKT3; eBioscience, San Diego, CA), IL-2 (50 IU/ml) (PeproTech), and different numbers of LAGE-1–specific CD4+ T cell clones. .. After 5 d, cells were collected and stained with anti–CD4-PE, anti–CD14-ECD, anti–CD19-ECD, and anti– CD8-PE-Cy5 (Beckman Coulter, Fullerton, CA), and proliferation of responder naive cells was analyzed by flow cytometry.

    Article Title: HIF-1α inhibitor echinomycin reduces acute graft-versus-host disease and preserves graft-versus-leukemia effect
    Article Snippet: After fixation and permeabilization, cells were stained with anti-mouse IL-17A-PE or anti-mouse IFN-γ PE antibodies. .. For T cell proliferation analysis purified CD4 T cells were stained with CFSE (5 µM; eBioscience, San Diego, CA, USA) and cultured alone or with allogeneic BMDCs for 5 days. .. To determine the apoptosis of A20 cells, Annexin V Apoptosis Detection Kit (eBioscience, San Diego, CA, USA) was used.

    Irradiation:

    Article Title: Epitope Hierarchy of Spontaneous CD4+ T Cell Responses to LAGE-1
    Article Snippet: Naive CD4+ and CD8+ T cells were purified from human PBMCs obtained from the blood of healthy donors by magnetic cell separation using CD45RA microbeads following the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA) and stained with CFSE (Invitrogen). .. CFSE-labeled naive T cells (1 × 105 ) were cultured for 5 d in 96-well flat bottom plates containing 2 × 105 irradiated (5000 rad) autologous CD3-depleted APCs, 0.1 µg/ml anti-CD3 (clone OKT3; eBioscience, San Diego, CA), IL-2 (50 IU/ml) (PeproTech), and different numbers of LAGE-1–specific CD4+ T cell clones. .. After 5 d, cells were collected and stained with anti–CD4-PE, anti–CD14-ECD, anti–CD19-ECD, and anti– CD8-PE-Cy5 (Beckman Coulter, Fullerton, CA), and proliferation of responder naive cells was analyzed by flow cytometry.

    Clone Assay:

    Article Title: Epitope Hierarchy of Spontaneous CD4+ T Cell Responses to LAGE-1
    Article Snippet: Naive CD4+ and CD8+ T cells were purified from human PBMCs obtained from the blood of healthy donors by magnetic cell separation using CD45RA microbeads following the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA) and stained with CFSE (Invitrogen). .. CFSE-labeled naive T cells (1 × 105 ) were cultured for 5 d in 96-well flat bottom plates containing 2 × 105 irradiated (5000 rad) autologous CD3-depleted APCs, 0.1 µg/ml anti-CD3 (clone OKT3; eBioscience, San Diego, CA), IL-2 (50 IU/ml) (PeproTech), and different numbers of LAGE-1–specific CD4+ T cell clones. .. After 5 d, cells were collected and stained with anti–CD4-PE, anti–CD14-ECD, anti–CD19-ECD, and anti– CD8-PE-Cy5 (Beckman Coulter, Fullerton, CA), and proliferation of responder naive cells was analyzed by flow cytometry.

    In Vitro:

    Article Title: Different populations of CD11b+ dendritic cells drive Th2 responses in the small intestine and colon
    Article Snippet: Cells were fixed and permeabilized using the eBioscience Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and intracellular staining was performed following the manufacturer's instructions. .. In vitro co-cultures For in vitro OT-II co-cultures 2 × 105 OT-II MLN cells were labelled with CFSE (eBioscience) at a dilution of 1:1,000 and cocultured with 6,000 sMLN or 3,000 cMLN FACS-sorted DCs from each population. ..

    FACS:

    Article Title: Different populations of CD11b+ dendritic cells drive Th2 responses in the small intestine and colon
    Article Snippet: Cells were fixed and permeabilized using the eBioscience Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and intracellular staining was performed following the manufacturer's instructions. .. In vitro co-cultures For in vitro OT-II co-cultures 2 × 105 OT-II MLN cells were labelled with CFSE (eBioscience) at a dilution of 1:1,000 and cocultured with 6,000 sMLN or 3,000 cMLN FACS-sorted DCs from each population. ..

    Flow Cytometry:

    Article Title: Host Immunity to Clostridium difficile PCR Ribotype 017 Strains
    Article Snippet: Next, splenocytes at 0.5 × 106 /ml (in PBS) were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; eBioscience) to a final concentration of 10 μM. .. Dynabeads mouse T-activator CD3/CD28 were added to CFSE-labeled splenocytes at a bead/cell ratio of 1:5 and then cocultured with C. difficile -stimulated BMDCs in a 96-well plate at a DC/splenocyte ratio of 1:10 at 37°C for 72 to 96 h. Cells were costained with anti-mouse CD4–PE-Cy5 (eBioscience) and analyzed by flow cytometry gated on CD4+ cells. .. Colonic pinch biopsy specimens from individuals ( n = 30; mean age, 10.4 ± 4.7 [the standard deviation]) undergoing routine endoscopy for gastrointestinal (GI) symptoms (e.g., constipation and allergy) were obtained.

    Article Title: Glycolytic metabolism is essential for CCR7 oligomerization and dendritic cell migration
    Article Snippet: After 45 h, the draining popliteal LNs were isolated (popliteal LNs from each mouse were pooled) and digested with DNase I (10 μg/mL) and collagenase D (1 mg/mL). .. CFSE+ cells in the dLNs were detected by flow cytometry and cells were counted using 123count eBeads (eBioscience). .. HDM-induced allergic asthma model: To investigate the effects of 2-DG on DC activation and migration, female Balb/c (7–8 weeks) were briefly anaesthetized with isoflurane and treated intranasally with 40 µg of low-endotoxin HDM (LE-HDM; Greer) in a volume of 30 µL.

    Article Title: Leptin Enhances Availability of Apoptotic Cell-Derived Self-Antigen in Systemic Lupus Erythematosus
    Article Snippet: To study OVA-TCR transgenic T cell responses to OVA-containing apoptotic bodies, OVA-TCR transgenic DO11.10 mice were immunized s.c. with 2 µg OVA (Sigma-Aldrich) emulsified 1∶1 in complete Freund adjuvant (CFA) (Sigma-Aldrich). .. After 72 h, mice were sacrificed, splenocytes stained with CFSE, and cocultured with TAMRA-labeled apoptotic bodies containing OVA (see above) in the presence of scalar doses of leptin or vehicle for 48 h before staining with clonotype-specific KJ1.26 Ab (eBioscience) and flow cytometry for enumeration of OVA-specific T cells. ..

    Cytometry:

    Article Title: Host Immunity to Clostridium difficile PCR Ribotype 017 Strains
    Article Snippet: Next, splenocytes at 0.5 × 106 /ml (in PBS) were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; eBioscience) to a final concentration of 10 μM. .. Dynabeads mouse T-activator CD3/CD28 were added to CFSE-labeled splenocytes at a bead/cell ratio of 1:5 and then cocultured with C. difficile -stimulated BMDCs in a 96-well plate at a DC/splenocyte ratio of 1:10 at 37°C for 72 to 96 h. Cells were costained with anti-mouse CD4–PE-Cy5 (eBioscience) and analyzed by flow cytometry gated on CD4+ cells. .. Colonic pinch biopsy specimens from individuals ( n = 30; mean age, 10.4 ± 4.7 [the standard deviation]) undergoing routine endoscopy for gastrointestinal (GI) symptoms (e.g., constipation and allergy) were obtained.

    Article Title: Glycolytic metabolism is essential for CCR7 oligomerization and dendritic cell migration
    Article Snippet: After 45 h, the draining popliteal LNs were isolated (popliteal LNs from each mouse were pooled) and digested with DNase I (10 μg/mL) and collagenase D (1 mg/mL). .. CFSE+ cells in the dLNs were detected by flow cytometry and cells were counted using 123count eBeads (eBioscience). .. HDM-induced allergic asthma model: To investigate the effects of 2-DG on DC activation and migration, female Balb/c (7–8 weeks) were briefly anaesthetized with isoflurane and treated intranasally with 40 µg of low-endotoxin HDM (LE-HDM; Greer) in a volume of 30 µL.

    Article Title: Leptin Enhances Availability of Apoptotic Cell-Derived Self-Antigen in Systemic Lupus Erythematosus
    Article Snippet: To study OVA-TCR transgenic T cell responses to OVA-containing apoptotic bodies, OVA-TCR transgenic DO11.10 mice were immunized s.c. with 2 µg OVA (Sigma-Aldrich) emulsified 1∶1 in complete Freund adjuvant (CFA) (Sigma-Aldrich). .. After 72 h, mice were sacrificed, splenocytes stained with CFSE, and cocultured with TAMRA-labeled apoptotic bodies containing OVA (see above) in the presence of scalar doses of leptin or vehicle for 48 h before staining with clonotype-specific KJ1.26 Ab (eBioscience) and flow cytometry for enumeration of OVA-specific T cells. ..

    Activation Assay:

    Article Title: Transiently Activated Human Regulatory T Cells Upregulate BCL-XL Expression and Acquire a Functional Advantage in vivo
    Article Snippet: In vivo Survival and Proliferation To measure in vivo survival and proliferation, freshly isolated Tregs were stained with 10 μM CTV (Invitrogen), activated for 15 h with anti-CD3/anti-CD28 beads (at a ratio of 1 bead to 5 cells) or left untreated. .. After activation, beads were carefully removed and 1 × 106 Treg cells were mixed with 5 × 106 PBMCs, labeled with 1 μM CFSE (eBioscience), from the same donor. .. PBMCs and Tregs were injected intraperitoneally into BALB/c Rag2−/− IL2rγ−/− mice and human cells isolated from peritoneal lavage on d5 for analysis as previously described ( ).

    Labeling:

    Article Title: Transiently Activated Human Regulatory T Cells Upregulate BCL-XL Expression and Acquire a Functional Advantage in vivo
    Article Snippet: In vivo Survival and Proliferation To measure in vivo survival and proliferation, freshly isolated Tregs were stained with 10 μM CTV (Invitrogen), activated for 15 h with anti-CD3/anti-CD28 beads (at a ratio of 1 bead to 5 cells) or left untreated. .. After activation, beads were carefully removed and 1 × 106 Treg cells were mixed with 5 × 106 PBMCs, labeled with 1 μM CFSE (eBioscience), from the same donor. .. PBMCs and Tregs were injected intraperitoneally into BALB/c Rag2−/− IL2rγ−/− mice and human cells isolated from peritoneal lavage on d5 for analysis as previously described ( ).

    Purification:

    Article Title: HIF-1α inhibitor echinomycin reduces acute graft-versus-host disease and preserves graft-versus-leukemia effect
    Article Snippet: After fixation and permeabilization, cells were stained with anti-mouse IL-17A-PE or anti-mouse IFN-γ PE antibodies. .. For T cell proliferation analysis purified CD4 T cells were stained with CFSE (5 µM; eBioscience, San Diego, CA, USA) and cultured alone or with allogeneic BMDCs for 5 days. .. To determine the apoptosis of A20 cells, Annexin V Apoptosis Detection Kit (eBioscience, San Diego, CA, USA) was used.

    Staining:

    Article Title: HIF-1α inhibitor echinomycin reduces acute graft-versus-host disease and preserves graft-versus-leukemia effect
    Article Snippet: After fixation and permeabilization, cells were stained with anti-mouse IL-17A-PE or anti-mouse IFN-γ PE antibodies. .. For T cell proliferation analysis purified CD4 T cells were stained with CFSE (5 µM; eBioscience, San Diego, CA, USA) and cultured alone or with allogeneic BMDCs for 5 days. .. To determine the apoptosis of A20 cells, Annexin V Apoptosis Detection Kit (eBioscience, San Diego, CA, USA) was used.

    Article Title: Leptin Enhances Availability of Apoptotic Cell-Derived Self-Antigen in Systemic Lupus Erythematosus
    Article Snippet: To study OVA-TCR transgenic T cell responses to OVA-containing apoptotic bodies, OVA-TCR transgenic DO11.10 mice were immunized s.c. with 2 µg OVA (Sigma-Aldrich) emulsified 1∶1 in complete Freund adjuvant (CFA) (Sigma-Aldrich). .. After 72 h, mice were sacrificed, splenocytes stained with CFSE, and cocultured with TAMRA-labeled apoptotic bodies containing OVA (see above) in the presence of scalar doses of leptin or vehicle for 48 h before staining with clonotype-specific KJ1.26 Ab (eBioscience) and flow cytometry for enumeration of OVA-specific T cells. ..

    Mouse Assay:

    Article Title: Leptin Enhances Availability of Apoptotic Cell-Derived Self-Antigen in Systemic Lupus Erythematosus
    Article Snippet: To study OVA-TCR transgenic T cell responses to OVA-containing apoptotic bodies, OVA-TCR transgenic DO11.10 mice were immunized s.c. with 2 µg OVA (Sigma-Aldrich) emulsified 1∶1 in complete Freund adjuvant (CFA) (Sigma-Aldrich). .. After 72 h, mice were sacrificed, splenocytes stained with CFSE, and cocultured with TAMRA-labeled apoptotic bodies containing OVA (see above) in the presence of scalar doses of leptin or vehicle for 48 h before staining with clonotype-specific KJ1.26 Ab (eBioscience) and flow cytometry for enumeration of OVA-specific T cells. ..

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    Thermo Fisher cfse
    The effect of LF on myeloid cells is mediated by the LRP2 receptor. ( A ) Expression of indicated LF receptors in MDSCs from 2-week-old mice measured by qRT-PCR ( n = 6). ( B ) Expression of Lrp2 in myeloid cells from mice of different ages measured by qRT-PCR ( n = 6). ( C ) Surface expression of Lrp2 on mouse myeloid cells measured by flow cytometry ( n = 6). ( D ) Expression of LF receptors in myeloid cells from PB (PBMC) or CB in full-term infants measured by qRT-PCR ( n = 5). ( E ) Expression of ITLN1 and LRP2 in myeloid cells from PB of adults (AD) and full-term, preterm, and NEC infants or CB measured by qRT-PCR ( n = 5–6). ( F – K ) Silencing of Itln1 and Lrp2 in newborn mouse BM cells with shRNA or scramble shRNA (control) lentiviruses, followed by treatment with PBS or LF for 2 days. ( F ) The absolute numbers of PMN-MDSCs and M-MDSCs measured by flow cytometry ( n = 4–6). ( G ) Suppressive activity of PMN-MDSCs. Effectors were OT-I CD8 + T cells stimulated with SIINFEKL. T cell proliferation was measured in triplicate by <t>CFSE</t> staining ( n = 3). Upper panel shows typical example. Lower panel shows cumulative results. ( H ) Suppressive activity of M-MDSCs. Effectors were CD4 + or CD8 + T cells stimulated with <t>CD3/CD28</t> antibodies ( n = 3). Left panel shows typical example. Right panel shows cumulative results. ( I ) The amount of S100A9 in PMN-MDSCs measured by ELISA. ( J ) The amount of PGE2 in PMN-MDSCs measured by ELISA. ( K ) The amounts of nitrites in M-MDSCs measured by the Nitrite Assay Kit. ( I – K , n = 6) Data represent individual results and mean ± SD. P values were calculated using 2-sided Student’ s t tests ( F – K ) or 1-way ANOVA followed by Tukey-Kramer multiple-comparisons test ( A – E ). * P
    Cfse, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher vybrant cfda se cell tracer
    Hypertrophy of human intestinal muscle cells is induced by synthetic MGF by an Erk-5-dependent mechanism. A : representative images of muscle cell hypertrophy induced by synthetic MGF but cell division induced by rhIGF-I. Hypertrophy was measured in the same groups of cells by confocal imaging of <t>Vybrant</t> <t>CFDA</t> SE cell tracer-loaded cells treated for 4 days with either synthetic MGF (100 nM) or rhIGF-I (100 nM). B : Z-stack analysis showing increased mean cell volume in cells treated with synthetic MGF (100 nM) but not rhIGF-I (100 nM). C : smooth muscle cell hypertrophy induced by synthetic MGF is inhibited by a selective Erk5 inhibitor. Hypertrophy was measured in cells incubated with synthetic MGF (100 nM) for 4 days in the presence or absence of BIX02188 (30 μM) from total cell protein per 10 4 cells. D : smooth muscle cell proliferation is induced by rhIGF-I but not synthetic MGF. Proliferation was measured by [ 3 H]thymidine incorporation into quiescent muscle cells incubated for 24 h with either synthetic MGF (1–100 nM) or rhIGF-I (1–100 nM). Results are means ± SE of 5–6 separate experiments. * P
    Vybrant Cfda Se Cell Tracer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The effect of LF on myeloid cells is mediated by the LRP2 receptor. ( A ) Expression of indicated LF receptors in MDSCs from 2-week-old mice measured by qRT-PCR ( n = 6). ( B ) Expression of Lrp2 in myeloid cells from mice of different ages measured by qRT-PCR ( n = 6). ( C ) Surface expression of Lrp2 on mouse myeloid cells measured by flow cytometry ( n = 6). ( D ) Expression of LF receptors in myeloid cells from PB (PBMC) or CB in full-term infants measured by qRT-PCR ( n = 5). ( E ) Expression of ITLN1 and LRP2 in myeloid cells from PB of adults (AD) and full-term, preterm, and NEC infants or CB measured by qRT-PCR ( n = 5–6). ( F – K ) Silencing of Itln1 and Lrp2 in newborn mouse BM cells with shRNA or scramble shRNA (control) lentiviruses, followed by treatment with PBS or LF for 2 days. ( F ) The absolute numbers of PMN-MDSCs and M-MDSCs measured by flow cytometry ( n = 4–6). ( G ) Suppressive activity of PMN-MDSCs. Effectors were OT-I CD8 + T cells stimulated with SIINFEKL. T cell proliferation was measured in triplicate by CFSE staining ( n = 3). Upper panel shows typical example. Lower panel shows cumulative results. ( H ) Suppressive activity of M-MDSCs. Effectors were CD4 + or CD8 + T cells stimulated with CD3/CD28 antibodies ( n = 3). Left panel shows typical example. Right panel shows cumulative results. ( I ) The amount of S100A9 in PMN-MDSCs measured by ELISA. ( J ) The amount of PGE2 in PMN-MDSCs measured by ELISA. ( K ) The amounts of nitrites in M-MDSCs measured by the Nitrite Assay Kit. ( I – K , n = 6) Data represent individual results and mean ± SD. P values were calculated using 2-sided Student’ s t tests ( F – K ) or 1-way ANOVA followed by Tukey-Kramer multiple-comparisons test ( A – E ). * P

    Journal: The Journal of Clinical Investigation

    Article Title: Lactoferrin-induced myeloid-derived suppressor cell therapy attenuates pathologic inflammatory conditions in newborn mice

    doi: 10.1172/JCI128164

    Figure Lengend Snippet: The effect of LF on myeloid cells is mediated by the LRP2 receptor. ( A ) Expression of indicated LF receptors in MDSCs from 2-week-old mice measured by qRT-PCR ( n = 6). ( B ) Expression of Lrp2 in myeloid cells from mice of different ages measured by qRT-PCR ( n = 6). ( C ) Surface expression of Lrp2 on mouse myeloid cells measured by flow cytometry ( n = 6). ( D ) Expression of LF receptors in myeloid cells from PB (PBMC) or CB in full-term infants measured by qRT-PCR ( n = 5). ( E ) Expression of ITLN1 and LRP2 in myeloid cells from PB of adults (AD) and full-term, preterm, and NEC infants or CB measured by qRT-PCR ( n = 5–6). ( F – K ) Silencing of Itln1 and Lrp2 in newborn mouse BM cells with shRNA or scramble shRNA (control) lentiviruses, followed by treatment with PBS or LF for 2 days. ( F ) The absolute numbers of PMN-MDSCs and M-MDSCs measured by flow cytometry ( n = 4–6). ( G ) Suppressive activity of PMN-MDSCs. Effectors were OT-I CD8 + T cells stimulated with SIINFEKL. T cell proliferation was measured in triplicate by CFSE staining ( n = 3). Upper panel shows typical example. Lower panel shows cumulative results. ( H ) Suppressive activity of M-MDSCs. Effectors were CD4 + or CD8 + T cells stimulated with CD3/CD28 antibodies ( n = 3). Left panel shows typical example. Right panel shows cumulative results. ( I ) The amount of S100A9 in PMN-MDSCs measured by ELISA. ( J ) The amount of PGE2 in PMN-MDSCs measured by ELISA. ( K ) The amounts of nitrites in M-MDSCs measured by the Nitrite Assay Kit. ( I – K , n = 6) Data represent individual results and mean ± SD. P values were calculated using 2-sided Student’ s t tests ( F – K ) or 1-way ANOVA followed by Tukey-Kramer multiple-comparisons test ( A – E ). * P

    Article Snippet: To evaluate M-MDSC–suppressive activity, sorted CD3+ T cells from spleen were labeled with CFSE (2 μM) (Invitrogen), stimulated with anti-CD3–coated (5 μg/mL) plates and soluble anti-CD28 (1 μg/mL) antibody (eBioscience), and cultured alone or with M-MDSCs at different ratios for 3 days.

    Techniques: Expressing, Mouse Assay, Quantitative RT-PCR, Flow Cytometry, Cytometry, shRNA, Activity Assay, Staining, Enzyme-linked Immunosorbent Assay, Nitration

    LF converts PMN and MON to MDSCs. ( A ) BM cells from 2-week-old mice were treated with LF or vehicle (PBS) in the presence of GM-CSF for 2 days. Typical example of flow cytometry (left), absolute numbers, and proportion of PMN and MON (right) ( n = 6). ( B ) Suppressive activity of LF-induced PMN-MDSCs against proliferation of OT-I–derived CD8 + T cells stimulated with cognate peptide ( n = 6). Nonstimulated T cells (No-stim) and stimulated T cells without PMN (No-PMN) were used as controls. ( C ) Absolute numbers of CD11b + HLA-DR –/lo cells in PBS- or LF-treated CB mononuclear cells for 2 days ( n = 6). ( D ) Suppression of LF-induced CD11b + HLA-DR –/lo cells from CB mononuclear cells against T cells stimulated with CD3/CD28 antibodies. T cell proliferation was measured in triplicate by CFSE dilution ( n = 6). ( E ) PGE2 and NO in myeloid cells from PBS- or LF-treated CB mononuclear cells ( n = 6). ( F ) Absolute numbers of myeloid cells in PBS- or LF-treated preterm infant–derived CB mononuclear cells for 2 days ( n = 6). ( G ) CD11b + HLA-DR –/lo were sorted from PBS- or LF-treated preterm infant CB mononuclear cells, followed by incubation with T cells stimulated with CD3/CD28 antibodies. T cell proliferation was measured in triplicate by CFSE labeling ( n = 6). In all plots, the results of individual experiments and mean ± SD are shown. P values were calculated using 2-sided Student’s t tests ( A and C – G ) or 1-way ANOVA followed by Tukey-Kramer multiple-comparisons test ( B ). * P

    Journal: The Journal of Clinical Investigation

    Article Title: Lactoferrin-induced myeloid-derived suppressor cell therapy attenuates pathologic inflammatory conditions in newborn mice

    doi: 10.1172/JCI128164

    Figure Lengend Snippet: LF converts PMN and MON to MDSCs. ( A ) BM cells from 2-week-old mice were treated with LF or vehicle (PBS) in the presence of GM-CSF for 2 days. Typical example of flow cytometry (left), absolute numbers, and proportion of PMN and MON (right) ( n = 6). ( B ) Suppressive activity of LF-induced PMN-MDSCs against proliferation of OT-I–derived CD8 + T cells stimulated with cognate peptide ( n = 6). Nonstimulated T cells (No-stim) and stimulated T cells without PMN (No-PMN) were used as controls. ( C ) Absolute numbers of CD11b + HLA-DR –/lo cells in PBS- or LF-treated CB mononuclear cells for 2 days ( n = 6). ( D ) Suppression of LF-induced CD11b + HLA-DR –/lo cells from CB mononuclear cells against T cells stimulated with CD3/CD28 antibodies. T cell proliferation was measured in triplicate by CFSE dilution ( n = 6). ( E ) PGE2 and NO in myeloid cells from PBS- or LF-treated CB mononuclear cells ( n = 6). ( F ) Absolute numbers of myeloid cells in PBS- or LF-treated preterm infant–derived CB mononuclear cells for 2 days ( n = 6). ( G ) CD11b + HLA-DR –/lo were sorted from PBS- or LF-treated preterm infant CB mononuclear cells, followed by incubation with T cells stimulated with CD3/CD28 antibodies. T cell proliferation was measured in triplicate by CFSE labeling ( n = 6). In all plots, the results of individual experiments and mean ± SD are shown. P values were calculated using 2-sided Student’s t tests ( A and C – G ) or 1-way ANOVA followed by Tukey-Kramer multiple-comparisons test ( B ). * P

    Article Snippet: To evaluate M-MDSC–suppressive activity, sorted CD3+ T cells from spleen were labeled with CFSE (2 μM) (Invitrogen), stimulated with anti-CD3–coated (5 μg/mL) plates and soluble anti-CD28 (1 μg/mL) antibody (eBioscience), and cultured alone or with M-MDSCs at different ratios for 3 days.

    Techniques: Mouse Assay, Flow Cytometry, Cytometry, Activity Assay, Incubation, Labeling

    LF regulates MDSC function via NF-κB transcription factor. ( A ) Western blot of p65 in cytoplasm (Cyt) and nuclei (Nuc) in MDSCs from newborn mouse BM cells treated with LF and/or NF-κB inhibitor (JSH23, 2.5 μM). Control cells were treated with PBS. Typical example of 3 performed experiments is shown. ( B ) Typical example of staining (on the left) and absolute numbers of PMN-MDSCs and M-MDSCs (right) from mouse BM cells treated with LF and/or JSH23 ( n = 6). ( C ) Suppressive activity of PMN-MDSCs ( n = 6). T cell proliferation of OT-I CD8 + T cells stimulated with SIINFEKL was measured in triplicate by CFSE staining. Mean ± SD are shown. ( D ) The amount of S100A9 in PMN-MDSCs measured by ELISA. ( E ) The amount of PGE2 in PMN-MDSCs measured by ELISA ( n = 6). ( F ) Absolute numbers of myeloid cells from LF-treated CB mononuclear cells in the presence or absence of JSH23 ( n = 6). ( G ) Suppressive activity of myeloid cells from LF-treated CB cells in the presence or absence of JSH23. Effectors were CD4 + or CD8 + T cells stimulated with CD3/CD28 antibodies. Proliferation was measured in triplicate by CFSE staining. Results of individual experiments are shown ( n = 4). In all plots, data represent mean ± SD. P values were calculated using 1-way ANOVA followed by Tukey-Kramer multiple-comparisons test. * P

    Journal: The Journal of Clinical Investigation

    Article Title: Lactoferrin-induced myeloid-derived suppressor cell therapy attenuates pathologic inflammatory conditions in newborn mice

    doi: 10.1172/JCI128164

    Figure Lengend Snippet: LF regulates MDSC function via NF-κB transcription factor. ( A ) Western blot of p65 in cytoplasm (Cyt) and nuclei (Nuc) in MDSCs from newborn mouse BM cells treated with LF and/or NF-κB inhibitor (JSH23, 2.5 μM). Control cells were treated with PBS. Typical example of 3 performed experiments is shown. ( B ) Typical example of staining (on the left) and absolute numbers of PMN-MDSCs and M-MDSCs (right) from mouse BM cells treated with LF and/or JSH23 ( n = 6). ( C ) Suppressive activity of PMN-MDSCs ( n = 6). T cell proliferation of OT-I CD8 + T cells stimulated with SIINFEKL was measured in triplicate by CFSE staining. Mean ± SD are shown. ( D ) The amount of S100A9 in PMN-MDSCs measured by ELISA. ( E ) The amount of PGE2 in PMN-MDSCs measured by ELISA ( n = 6). ( F ) Absolute numbers of myeloid cells from LF-treated CB mononuclear cells in the presence or absence of JSH23 ( n = 6). ( G ) Suppressive activity of myeloid cells from LF-treated CB cells in the presence or absence of JSH23. Effectors were CD4 + or CD8 + T cells stimulated with CD3/CD28 antibodies. Proliferation was measured in triplicate by CFSE staining. Results of individual experiments are shown ( n = 4). In all plots, data represent mean ± SD. P values were calculated using 1-way ANOVA followed by Tukey-Kramer multiple-comparisons test. * P

    Article Snippet: To evaluate M-MDSC–suppressive activity, sorted CD3+ T cells from spleen were labeled with CFSE (2 μM) (Invitrogen), stimulated with anti-CD3–coated (5 μg/mL) plates and soluble anti-CD28 (1 μg/mL) antibody (eBioscience), and cultured alone or with M-MDSCs at different ratios for 3 days.

    Techniques: Western Blot, Staining, Activity Assay, Enzyme-linked Immunosorbent Assay

    Clinical significance of MDSCs in infants. ( A ) Percentages of Lox1 + CD15 + PMN-MDSCs in adults (AD) ( n = 10), full-term (term) ( n = 8), and preterm ( n = 11) infants and patients with NEC ( n = 14). Samples from NEC patients were collected at diagnosis (6 to 66 days after birth). In all other cases, samples were collected between days 1 and 4 after birth. ( B ) Percentages of PMN-MDSCs in infants who later developed or did not develop NEC. Samples were collected on days 1–3 (AD, n = 10; term, n = 8; preterm, no NEC, n = 51; preterm with NEC, n = 9) or 4–7 (term, n = 9; preterm, no NEC, n = 15; preterm with NEC, n = 5) after birth. ( C ) Correlation between proportion of PMN-MDSCs and body weight of infants at different age. ( D ) Functional activity of PMN-MDSCs from infants using T cells stimulated with CD3/CD28 antibodies. T cell proliferation was measured in triplicate with CFSE labeling ( n = 4–8). ( E ) Expression of indicated genes in PMN-MDSCs measured by quantitative reverse-transcriptase PCR (qRT-PCR) ( n = 4). ( F ) Antibacterial activity of PMN-MDSCs against E . coli ( n = 4). ( G ) Concentrations of LF in plasma from adults and newborns determined by ELISA ( n = 8–11). ( H ) Correlation between LF concentration (plasma) and proportion of PMN-MDSCs, body weight, or gestational age of newborns. ( I ) LF concentration in plasma of infants who were fed with breast milk ( n = 13) or formula ( n = 4). In all panels, individual results and mean ± SD are shown. P values were calculated using 2-sided Student’s t tests ( E , F , and I ) or 1-way ANOVA followed by Tukey-Kramer multiple-comparisons test ( A , B , D , and G ). * P

    Journal: The Journal of Clinical Investigation

    Article Title: Lactoferrin-induced myeloid-derived suppressor cell therapy attenuates pathologic inflammatory conditions in newborn mice

    doi: 10.1172/JCI128164

    Figure Lengend Snippet: Clinical significance of MDSCs in infants. ( A ) Percentages of Lox1 + CD15 + PMN-MDSCs in adults (AD) ( n = 10), full-term (term) ( n = 8), and preterm ( n = 11) infants and patients with NEC ( n = 14). Samples from NEC patients were collected at diagnosis (6 to 66 days after birth). In all other cases, samples were collected between days 1 and 4 after birth. ( B ) Percentages of PMN-MDSCs in infants who later developed or did not develop NEC. Samples were collected on days 1–3 (AD, n = 10; term, n = 8; preterm, no NEC, n = 51; preterm with NEC, n = 9) or 4–7 (term, n = 9; preterm, no NEC, n = 15; preterm with NEC, n = 5) after birth. ( C ) Correlation between proportion of PMN-MDSCs and body weight of infants at different age. ( D ) Functional activity of PMN-MDSCs from infants using T cells stimulated with CD3/CD28 antibodies. T cell proliferation was measured in triplicate with CFSE labeling ( n = 4–8). ( E ) Expression of indicated genes in PMN-MDSCs measured by quantitative reverse-transcriptase PCR (qRT-PCR) ( n = 4). ( F ) Antibacterial activity of PMN-MDSCs against E . coli ( n = 4). ( G ) Concentrations of LF in plasma from adults and newborns determined by ELISA ( n = 8–11). ( H ) Correlation between LF concentration (plasma) and proportion of PMN-MDSCs, body weight, or gestational age of newborns. ( I ) LF concentration in plasma of infants who were fed with breast milk ( n = 13) or formula ( n = 4). In all panels, individual results and mean ± SD are shown. P values were calculated using 2-sided Student’s t tests ( E , F , and I ) or 1-way ANOVA followed by Tukey-Kramer multiple-comparisons test ( A , B , D , and G ). * P

    Article Snippet: To evaluate M-MDSC–suppressive activity, sorted CD3+ T cells from spleen were labeled with CFSE (2 μM) (Invitrogen), stimulated with anti-CD3–coated (5 μg/mL) plates and soluble anti-CD28 (1 μg/mL) antibody (eBioscience), and cultured alone or with M-MDSCs at different ratios for 3 days.

    Techniques: Functional Assay, Activity Assay, Labeling, Expressing, Polymerase Chain Reaction, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Concentration Assay

    Lack of LDH-A in Mø induces CD8 + T cell activation A–B . CFSE based proliferation of T cells subsets was measured by flow cytometry after 48 hours co-culture of CD3 + T cells and splenocytes in the presence of LPS. The ratio of CFSE(diluted):CFSE(undiluted) CD8+ T cells is shown in B. C–D . CFSE based proliferation of wild type CD8 + T cells subset was measured by flow cytometry after 48 hours co-culture of CD8 + T cells and splenocytes isolated from wt ( LDH-A +/+ ) or ko ( LDH-A −/− ) mice. The ratio of CFSE(diluted):CFSE(undiluted) CD8+ T cells is shown in D. E–F . Real time PCR of IL-17 or IL-10 mRNA expression in CD8 + T cells MLR as in C . *p

    Journal: Cancer research

    Article Title: Deletion of lactate dehydrogenase-A in myeloid cells triggers antitumor immunity

    doi: 10.1158/0008-5472.CAN-16-2938

    Figure Lengend Snippet: Lack of LDH-A in Mø induces CD8 + T cell activation A–B . CFSE based proliferation of T cells subsets was measured by flow cytometry after 48 hours co-culture of CD3 + T cells and splenocytes in the presence of LPS. The ratio of CFSE(diluted):CFSE(undiluted) CD8+ T cells is shown in B. C–D . CFSE based proliferation of wild type CD8 + T cells subset was measured by flow cytometry after 48 hours co-culture of CD8 + T cells and splenocytes isolated from wt ( LDH-A +/+ ) or ko ( LDH-A −/− ) mice. The ratio of CFSE(diluted):CFSE(undiluted) CD8+ T cells is shown in D. E–F . Real time PCR of IL-17 or IL-10 mRNA expression in CD8 + T cells MLR as in C . *p

    Article Snippet: CD3+ cells were stained with CFSE (Molecular Probes, Invitrogen) following manufacturers’ protocol.

    Techniques: Activation Assay, Flow Cytometry, Cytometry, Co-Culture Assay, Isolation, Mouse Assay, Real-time Polymerase Chain Reaction, Expressing

    Schematic of the experimental workflow. We collected four types of data from each individual: 1) quality-controlled genome-wide SNP data containing 638,347 markers collected on Illumina Infinium Human OmniExpress Exome BeadChips, 2) abundance of CD4 T EM cells as a percentage of all CD4 T cells obtained by FACS and quantified by X-Cyt, 3) average cell division upon T cell receptor stimulation by anti-CD3/CD28 commercial beads, measured using a CFSE (carboxyfluorescein succinimidyl ester) dye dilution assay, and 4) expression of 215 genes measured by NanoString nCounter. We repeated each proliferation assay in two-three technical replicates. Cell sorting purity and replication correlations for CD4 T EM abundance, division index, and proliferation index are shown in Figure S2 .

    Journal: PLoS Genetics

    Article Title: Regulation of Gene Expression in Autoimmune Disease Loci and the Genetic Basis of Proliferation in CD4+ Effector Memory T Cells

    doi: 10.1371/journal.pgen.1004404

    Figure Lengend Snippet: Schematic of the experimental workflow. We collected four types of data from each individual: 1) quality-controlled genome-wide SNP data containing 638,347 markers collected on Illumina Infinium Human OmniExpress Exome BeadChips, 2) abundance of CD4 T EM cells as a percentage of all CD4 T cells obtained by FACS and quantified by X-Cyt, 3) average cell division upon T cell receptor stimulation by anti-CD3/CD28 commercial beads, measured using a CFSE (carboxyfluorescein succinimidyl ester) dye dilution assay, and 4) expression of 215 genes measured by NanoString nCounter. We repeated each proliferation assay in two-three technical replicates. Cell sorting purity and replication correlations for CD4 T EM abundance, division index, and proliferation index are shown in Figure S2 .

    Article Snippet: For proliferation studies, we labeled cells with carboxyfluorescein diacetate succinimidyl ester (CFSE; eBioscience), and measured proliferation by dye dilution.

    Techniques: Genome Wide, FACS, Dilution Assay, Expressing, Proliferation Assay

    Hypertrophy of human intestinal muscle cells is induced by synthetic MGF by an Erk-5-dependent mechanism. A : representative images of muscle cell hypertrophy induced by synthetic MGF but cell division induced by rhIGF-I. Hypertrophy was measured in the same groups of cells by confocal imaging of Vybrant CFDA SE cell tracer-loaded cells treated for 4 days with either synthetic MGF (100 nM) or rhIGF-I (100 nM). B : Z-stack analysis showing increased mean cell volume in cells treated with synthetic MGF (100 nM) but not rhIGF-I (100 nM). C : smooth muscle cell hypertrophy induced by synthetic MGF is inhibited by a selective Erk5 inhibitor. Hypertrophy was measured in cells incubated with synthetic MGF (100 nM) for 4 days in the presence or absence of BIX02188 (30 μM) from total cell protein per 10 4 cells. D : smooth muscle cell proliferation is induced by rhIGF-I but not synthetic MGF. Proliferation was measured by [ 3 H]thymidine incorporation into quiescent muscle cells incubated for 24 h with either synthetic MGF (1–100 nM) or rhIGF-I (1–100 nM). Results are means ± SE of 5–6 separate experiments. * P

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: Increased IGF-IEc expression and mechano-growth factor production in intestinal muscle of fibrostenotic Crohn's disease and smooth muscle hypertrophy

    doi: 10.1152/ajpgi.00414.2014

    Figure Lengend Snippet: Hypertrophy of human intestinal muscle cells is induced by synthetic MGF by an Erk-5-dependent mechanism. A : representative images of muscle cell hypertrophy induced by synthetic MGF but cell division induced by rhIGF-I. Hypertrophy was measured in the same groups of cells by confocal imaging of Vybrant CFDA SE cell tracer-loaded cells treated for 4 days with either synthetic MGF (100 nM) or rhIGF-I (100 nM). B : Z-stack analysis showing increased mean cell volume in cells treated with synthetic MGF (100 nM) but not rhIGF-I (100 nM). C : smooth muscle cell hypertrophy induced by synthetic MGF is inhibited by a selective Erk5 inhibitor. Hypertrophy was measured in cells incubated with synthetic MGF (100 nM) for 4 days in the presence or absence of BIX02188 (30 μM) from total cell protein per 10 4 cells. D : smooth muscle cell proliferation is induced by rhIGF-I but not synthetic MGF. Proliferation was measured by [ 3 H]thymidine incorporation into quiescent muscle cells incubated for 24 h with either synthetic MGF (1–100 nM) or rhIGF-I (1–100 nM). Results are means ± SE of 5–6 separate experiments. * P

    Article Snippet: Smooth muscle cell hypertrophy was measured in cells cultured on registered coverslip slides and microscope stage and loaded with Vybrant CFDA SE cell tracer (Life Technologies, Grand Island, NY).

    Techniques: Imaging, Incubation