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CD4 expression in cells used in the study. The OKT4 anti-CD4 monoclonal antibody was used to stain the surface of Jurkat T lymphocytes, whole human PBMC isolated by standard <t>Ficoll-Paque</t> density centrifugation, and a PBMC preparation enriched for CD4 + T cells. The staining of 293T cells, which were used as a negative control, is shown in gray.
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1) Product Images from "Importance of Membrane Fusion Mediated by Human Immunodeficiency Virus Envelope Glycoproteins for Lysis of Primary CD4-Positive T Cells"

Article Title: Importance of Membrane Fusion Mediated by Human Immunodeficiency Virus Envelope Glycoproteins for Lysis of Primary CD4-Positive T Cells

Journal: Journal of Virology

doi:

CD4 expression in cells used in the study. The OKT4 anti-CD4 monoclonal antibody was used to stain the surface of Jurkat T lymphocytes, whole human PBMC isolated by standard Ficoll-Paque density centrifugation, and a PBMC preparation enriched for CD4 + T cells. The staining of 293T cells, which were used as a negative control, is shown in gray.
Figure Legend Snippet: CD4 expression in cells used in the study. The OKT4 anti-CD4 monoclonal antibody was used to stain the surface of Jurkat T lymphocytes, whole human PBMC isolated by standard Ficoll-Paque density centrifugation, and a PBMC preparation enriched for CD4 + T cells. The staining of 293T cells, which were used as a negative control, is shown in gray.

Techniques Used: Expressing, Staining, Isolation, Centrifugation, Negative Control

2) Product Images from "A robust intracellular metabolite extraction protocol for human neutrophil metabolic profiling"

Article Title: A robust intracellular metabolite extraction protocol for human neutrophil metabolic profiling

Journal: PLoS ONE

doi: 10.1371/journal.pone.0209270

Optimisation of neutrophil isolation protocols for NMR metabolomics. (A) Spectra (n = 3) shown for neutrophil isolation protocols including (grey spectra) or omitting (red spectra) a wash step immediately after Ficoll-Paque separation. Metabolite loss in the washed samples is indicated by arrows. (B) Spectra (n = 3) show neutrophils incubated with media containing phosphate buffer (red spectra) or HEPES buffer (grey spectra). Multiple contaminating peaks from HEPES are indicated by arrows. (C) Spectra (n = 3) are shown for sample preparation including (grey spectra) or excluding (red spectra) a heat-shock step immediately prior to snap freezing of pellet. Metabolite degradation/turnover was prevented by heat shock as indicated by arrows.
Figure Legend Snippet: Optimisation of neutrophil isolation protocols for NMR metabolomics. (A) Spectra (n = 3) shown for neutrophil isolation protocols including (grey spectra) or omitting (red spectra) a wash step immediately after Ficoll-Paque separation. Metabolite loss in the washed samples is indicated by arrows. (B) Spectra (n = 3) show neutrophils incubated with media containing phosphate buffer (red spectra) or HEPES buffer (grey spectra). Multiple contaminating peaks from HEPES are indicated by arrows. (C) Spectra (n = 3) are shown for sample preparation including (grey spectra) or excluding (red spectra) a heat-shock step immediately prior to snap freezing of pellet. Metabolite degradation/turnover was prevented by heat shock as indicated by arrows.

Techniques Used: Isolation, Nuclear Magnetic Resonance, Incubation, Sample Prep

3) Product Images from "Six‐gene Assay as a new biomarker in the blood of patients with colorectal cancer: establishment and clinical validation"

Article Title: Six‐gene Assay as a new biomarker in the blood of patients with colorectal cancer: establishment and clinical validation

Journal: Molecular Oncology

doi: 10.1002/1878-0261.12427

Evaluation of cell detection efficiency of CEA Gene Assay and Six‐gene Assay. A dilution series of cells (1, 10, 100 and 1000) from CRC cell lines SW 1116 and SW 48 were respectively spiked in 1.0 mL of peripheral blood from a healthy donor. Blood samples were further processed by Ficoll‐Paque gradient separation, RNA extraction and real‐time quantitative PCR . The plot represents number of cells spiked versus number of cells observed. The recovery of spiked numbers of CRC cells was measured by CEA Gene Assay and Six‐gene Assay based on the mRNA expression of corresponding genes in CRC cell lines. Each error bar represents mean ± SD . Inset tables provide detailed numbers for each dilution.
Figure Legend Snippet: Evaluation of cell detection efficiency of CEA Gene Assay and Six‐gene Assay. A dilution series of cells (1, 10, 100 and 1000) from CRC cell lines SW 1116 and SW 48 were respectively spiked in 1.0 mL of peripheral blood from a healthy donor. Blood samples were further processed by Ficoll‐Paque gradient separation, RNA extraction and real‐time quantitative PCR . The plot represents number of cells spiked versus number of cells observed. The recovery of spiked numbers of CRC cells was measured by CEA Gene Assay and Six‐gene Assay based on the mRNA expression of corresponding genes in CRC cell lines. Each error bar represents mean ± SD . Inset tables provide detailed numbers for each dilution.

Techniques Used: Gene Assay, RNA Extraction, Real-time Polymerase Chain Reaction, Expressing

4) Product Images from "A feline CD2 homologue interacts with human red blood cells"

Article Title: A feline CD2 homologue interacts with human red blood cells

Journal: Immunology

doi: 10.1046/j.0019-2805.2001.01371.x

Flow cytometry (FCM) analysis of feline peripheral blood mononuclear cells (fPBMCs). fPBMCs expressing the feline homologue of CD2 (fCD2) (b) were gated and their light scatters are shown: (a) all fPBMCs isolated by Ficoll-Paque™; (c) fPBMCs strongly positive for fCD2; and (d) fPBMCs weakly positive for fCD2.
Figure Legend Snippet: Flow cytometry (FCM) analysis of feline peripheral blood mononuclear cells (fPBMCs). fPBMCs expressing the feline homologue of CD2 (fCD2) (b) were gated and their light scatters are shown: (a) all fPBMCs isolated by Ficoll-Paque™; (c) fPBMCs strongly positive for fCD2; and (d) fPBMCs weakly positive for fCD2.

Techniques Used: Flow Cytometry, Cytometry, Expressing, Isolation

5) Product Images from "A robust intracellular metabolite extraction protocol for human neutrophil metabolic profiling"

Article Title: A robust intracellular metabolite extraction protocol for human neutrophil metabolic profiling

Journal: PLoS ONE

doi: 10.1371/journal.pone.0209270

Optimisation of neutrophil isolation protocols for NMR metabolomics. (A) Spectra (n = 3) shown for neutrophil isolation protocols including (grey spectra) or omitting (red spectra) a wash step immediately after Ficoll-Paque separation. Metabolite loss in the washed samples is indicated by arrows. (B) Spectra (n = 3) show neutrophils incubated with media containing phosphate buffer (red spectra) or HEPES buffer (grey spectra). Multiple contaminating peaks from HEPES are indicated by arrows. (C) Spectra (n = 3) are shown for sample preparation including (grey spectra) or excluding (red spectra) a heat-shock step immediately prior to snap freezing of pellet. Metabolite degradation/turnover was prevented by heat shock as indicated by arrows.
Figure Legend Snippet: Optimisation of neutrophil isolation protocols for NMR metabolomics. (A) Spectra (n = 3) shown for neutrophil isolation protocols including (grey spectra) or omitting (red spectra) a wash step immediately after Ficoll-Paque separation. Metabolite loss in the washed samples is indicated by arrows. (B) Spectra (n = 3) show neutrophils incubated with media containing phosphate buffer (red spectra) or HEPES buffer (grey spectra). Multiple contaminating peaks from HEPES are indicated by arrows. (C) Spectra (n = 3) are shown for sample preparation including (grey spectra) or excluding (red spectra) a heat-shock step immediately prior to snap freezing of pellet. Metabolite degradation/turnover was prevented by heat shock as indicated by arrows.

Techniques Used: Isolation, Nuclear Magnetic Resonance, Incubation, Sample Prep

6) Product Images from "Glucocorticoid-induced TNF receptor-triggered T cells are key modulators for survival/death of neural stem/progenitor cells induced by ischemic stroke"

Article Title: Glucocorticoid-induced TNF receptor-triggered T cells are key modulators for survival/death of neural stem/progenitor cells induced by ischemic stroke

Journal: Cell Death and Differentiation

doi: 10.1038/cdd.2011.145

The analysis for the subpopulation of infiltrated cells in the ischemic area with FACS. ( a ) The ischemic tissue of the brain 7 days or 1 day after stroke was isolated and pressed through a cell strainer, and was separated by Ficoll-paque plus centrifugation. The extract contains lymphocyte-like mononuclear cells, which were observed under phase-contrast microscope. ( b ) FACS analysis for the subset of T cells that infiltrated the ischemic cortex was performed. The acquired lymphocytes were analyzed for CD4 + and CD25 + on CD3 + cells 1 day ( c ) and 7 days ( e ), and for GITR on CD3 + CD4 + cells 1 day ( d ) or 7 days ( f ) after stroke. The percentage of CD25 + cells was evaluated in the total T cells (CD3 + cells), and that of GITR + cells was evaluated in CD4 + T (CD3 + CD4 + ) cells extracted from the infarcted brain tissue. The mean channel values were displayed for GITR in the CD3 + CD4 + cells. ( d and f ) Filled histogram represents GITR expression and open histogram represents isotype control
Figure Legend Snippet: The analysis for the subpopulation of infiltrated cells in the ischemic area with FACS. ( a ) The ischemic tissue of the brain 7 days or 1 day after stroke was isolated and pressed through a cell strainer, and was separated by Ficoll-paque plus centrifugation. The extract contains lymphocyte-like mononuclear cells, which were observed under phase-contrast microscope. ( b ) FACS analysis for the subset of T cells that infiltrated the ischemic cortex was performed. The acquired lymphocytes were analyzed for CD4 + and CD25 + on CD3 + cells 1 day ( c ) and 7 days ( e ), and for GITR on CD3 + CD4 + cells 1 day ( d ) or 7 days ( f ) after stroke. The percentage of CD25 + cells was evaluated in the total T cells (CD3 + cells), and that of GITR + cells was evaluated in CD4 + T (CD3 + CD4 + ) cells extracted from the infarcted brain tissue. The mean channel values were displayed for GITR in the CD3 + CD4 + cells. ( d and f ) Filled histogram represents GITR expression and open histogram represents isotype control

Techniques Used: FACS, Isolation, Centrifugation, Microscopy, Expressing

7) Product Images from "Envelope Is a Major Viral Determinant of the Distinct In Vitro Cellular Transformation Tropism of Human T-Cell Leukemia Virus Type 1 (HTLV-1) and HTLV-2"

Article Title: Envelope Is a Major Viral Determinant of the Distinct In Vitro Cellular Transformation Tropism of Human T-Cell Leukemia Virus Type 1 (HTLV-1) and HTLV-2

Journal: Journal of Virology

doi: 10.1128/JVI.79.23.14536-14545.2005

Growth curve for HTLV T-lymphocyte transformation assay. Human PBMCs were isolated by Ficoll-Paque and cocultivated with irradiated (10,000 rads) 729 producer cells (729-wtHTLV-1, 729-wtHTLV-2, 729-HTLV-2/LTR1, 729-HTLV-1/LTR2, 729-HTLV-1/Env2, or 729-HTLV-2/Env1) or 729 uninfected control cells as indicated. PBMCs (2 × 10 6 ) were cultured with donor cells (1 × 10 6 ) in 24-well plates. Cells were fed once per week with medium containing 20% FBS. (A) Cell viability was determined weekly by trypan blue exclusion from 0 to 8 weeks postcocultivation. The mean and standard deviation for each time point were determined from three independent samples. (B) The presence of HTLV gene expression was confirmed by detection of structural Gag protein in the culture supernatant by p19 ELISA at 3, 4, 5, 6, 7, and 8 weeks postcocultivation. The mean and standard deviation for each time point were determined from three independent samples.
Figure Legend Snippet: Growth curve for HTLV T-lymphocyte transformation assay. Human PBMCs were isolated by Ficoll-Paque and cocultivated with irradiated (10,000 rads) 729 producer cells (729-wtHTLV-1, 729-wtHTLV-2, 729-HTLV-2/LTR1, 729-HTLV-1/LTR2, 729-HTLV-1/Env2, or 729-HTLV-2/Env1) or 729 uninfected control cells as indicated. PBMCs (2 × 10 6 ) were cultured with donor cells (1 × 10 6 ) in 24-well plates. Cells were fed once per week with medium containing 20% FBS. (A) Cell viability was determined weekly by trypan blue exclusion from 0 to 8 weeks postcocultivation. The mean and standard deviation for each time point were determined from three independent samples. (B) The presence of HTLV gene expression was confirmed by detection of structural Gag protein in the culture supernatant by p19 ELISA at 3, 4, 5, 6, 7, and 8 weeks postcocultivation. The mean and standard deviation for each time point were determined from three independent samples.

Techniques Used: Transformation Assay, Isolation, Irradiation, Cell Culture, Standard Deviation, Expressing, Enzyme-linked Immunosorbent Assay

8) Product Images from "Direct measurement of lymphocyte receptor diversity"

Article Title: Direct measurement of lymphocyte receptor diversity

Journal: Nucleic Acids Research

doi: 10.1093/nar/gng139

Analysis of B cell diversity using the gene chip method. Splenocytes were harvested from 3- to 4-week-old J H –/– , QM and WT mice and mononuclear cells were isolated on Ficoll-paque gradients. Total RNA was isolated from the lymphocytes and first-strand cDNA was generated using a primer designed to bind the constant region of the mouse heavy chain J H4 region plus the T7 polymerase promoter. The custom primer promoted amplification of J H4 heavy chain-specific RNA only. Equal amounts of the in vitro transcription product (cRNA) from each mouse and standards were hybridized to gene chips and then the chips were stained and analyzed as described in Materials and Methods. ( A ) B cell heavy chain diversity in mutant mice before and after immunization with KLH. Pre-immunization, WT diversity (filled circle) was more than 2-fold higher than QM (open circle) diversity. Post-immunization, WT diversity (filled circle) decreased (4.0 × 10 4 different B cell heavy chain clones) while QM (open circle) diversity increased (7.5 × 10 3 ). Background hybridization was established using J H –/– RNA (filled square). ( B ) Immune responsiveness to KLH. An ELISA was used to detect levels of anti-KLH antibodies in the serum following immunization. The QM anti-KLH antibody titer was ∼40% of the wild-type following immunization. *P
Figure Legend Snippet: Analysis of B cell diversity using the gene chip method. Splenocytes were harvested from 3- to 4-week-old J H –/– , QM and WT mice and mononuclear cells were isolated on Ficoll-paque gradients. Total RNA was isolated from the lymphocytes and first-strand cDNA was generated using a primer designed to bind the constant region of the mouse heavy chain J H4 region plus the T7 polymerase promoter. The custom primer promoted amplification of J H4 heavy chain-specific RNA only. Equal amounts of the in vitro transcription product (cRNA) from each mouse and standards were hybridized to gene chips and then the chips were stained and analyzed as described in Materials and Methods. ( A ) B cell heavy chain diversity in mutant mice before and after immunization with KLH. Pre-immunization, WT diversity (filled circle) was more than 2-fold higher than QM (open circle) diversity. Post-immunization, WT diversity (filled circle) decreased (4.0 × 10 4 different B cell heavy chain clones) while QM (open circle) diversity increased (7.5 × 10 3 ). Background hybridization was established using J H –/– RNA (filled square). ( B ) Immune responsiveness to KLH. An ELISA was used to detect levels of anti-KLH antibodies in the serum following immunization. The QM anti-KLH antibody titer was ∼40% of the wild-type following immunization. *P

Techniques Used: Chromatin Immunoprecipitation, Mouse Assay, Isolation, Generated, Amplification, In Vitro, Staining, Mutagenesis, Hybridization, Enzyme-linked Immunosorbent Assay

9) Product Images from "A robust intracellular metabolite extraction protocol for human neutrophil metabolic profiling"

Article Title: A robust intracellular metabolite extraction protocol for human neutrophil metabolic profiling

Journal: PLoS ONE

doi: 10.1371/journal.pone.0209270

Optimisation of neutrophil isolation protocols for NMR metabolomics. (A) Spectra (n = 3) shown for neutrophil isolation protocols including (grey spectra) or omitting (red spectra) a wash step immediately after Ficoll-Paque separation. Metabolite loss in the washed samples is indicated by arrows. (B) Spectra (n = 3) show neutrophils incubated with media containing phosphate buffer (red spectra) or HEPES buffer (grey spectra). Multiple contaminating peaks from HEPES are indicated by arrows. (C) Spectra (n = 3) are shown for sample preparation including (grey spectra) or excluding (red spectra) a heat-shock step immediately prior to snap freezing of pellet. Metabolite degradation/turnover was prevented by heat shock as indicated by arrows.
Figure Legend Snippet: Optimisation of neutrophil isolation protocols for NMR metabolomics. (A) Spectra (n = 3) shown for neutrophil isolation protocols including (grey spectra) or omitting (red spectra) a wash step immediately after Ficoll-Paque separation. Metabolite loss in the washed samples is indicated by arrows. (B) Spectra (n = 3) show neutrophils incubated with media containing phosphate buffer (red spectra) or HEPES buffer (grey spectra). Multiple contaminating peaks from HEPES are indicated by arrows. (C) Spectra (n = 3) are shown for sample preparation including (grey spectra) or excluding (red spectra) a heat-shock step immediately prior to snap freezing of pellet. Metabolite degradation/turnover was prevented by heat shock as indicated by arrows.

Techniques Used: Isolation, Nuclear Magnetic Resonance, Incubation, Sample Prep

10) Product Images from "Local and systemic immunomodulatory mechanisms triggered by Human Papillomavirus transformed cells: a potential role for G-CSF and neutrophils"

Article Title: Local and systemic immunomodulatory mechanisms triggered by Human Papillomavirus transformed cells: a potential role for G-CSF and neutrophils

Journal: Scientific Reports

doi: 10.1038/s41598-017-09079-3

Cervical lesion systemic effects. ( A ) G-CSF plasma concentrations, measured by ELISA. We used plasma from 8 control donors, from 10 CC patients, 8 CIN1, 11 CIN2, 18 CIN3 and 17 ICC patients. Results were compared by one-way ANOVA, significant differences are indicated by *. ( B ) Frequency of immature circulating neutrophils. The mononuclear cells from the Ficoll-Paque gradient were harvested and labeled with anti-CD66b and CD11b. The graph shows the frequency of CD66b + CD11b − cells in each experimental group, which contained 5 controls, 4 CC patients, 13 high grade lesion patients (HG) and 11 ICC patients. We had only 2 patients with CIN1 lesions, therefore, had to exclude this group from this analysis. Again, results were tested with one-way ANOVA, and the p value is indicated. ( C ) Correlation between G-CSF plasma concentration and frequency of circulating immature neutrophils CD66b + . We used Pearson correlation to compare these parameters, R values indicated in each graph. ( D ) Allogeneic T cell stimulation. Cell proliferation dye labeled T cells from control donors were incubated for 4 days with MoDCs from patients of the indicated groups. After incubation, cells were harvested, labeled with CD4, CD8 and CD25 and analyzed by flow cytometry. Cell proliferation data shows the frequency of cells within the CD4 + or CD8 + T populations that diluted the proliferation dye, therefore, that proliferated during the experiments. CD25 expression, represented as median fluorescence intensity (MFI) was gated on CD4 T cells. We used one-way ANOVA to compare experimental groups. In all cases, samples from ICC patients were significantly different from the other patients. CC – cervicitis, LG – cells from patients with low grade lesions (or CIN1), HG – cells from patients with high grade lesions (CIN2/3), ICC – cells from patients with invasive cervical carcinoma. ( E ) Pearson correlations between T cell proliferation determined in D and plasma G-CSF concentrations, determined in B; R and p values are indicated in the plots.
Figure Legend Snippet: Cervical lesion systemic effects. ( A ) G-CSF plasma concentrations, measured by ELISA. We used plasma from 8 control donors, from 10 CC patients, 8 CIN1, 11 CIN2, 18 CIN3 and 17 ICC patients. Results were compared by one-way ANOVA, significant differences are indicated by *. ( B ) Frequency of immature circulating neutrophils. The mononuclear cells from the Ficoll-Paque gradient were harvested and labeled with anti-CD66b and CD11b. The graph shows the frequency of CD66b + CD11b − cells in each experimental group, which contained 5 controls, 4 CC patients, 13 high grade lesion patients (HG) and 11 ICC patients. We had only 2 patients with CIN1 lesions, therefore, had to exclude this group from this analysis. Again, results were tested with one-way ANOVA, and the p value is indicated. ( C ) Correlation between G-CSF plasma concentration and frequency of circulating immature neutrophils CD66b + . We used Pearson correlation to compare these parameters, R values indicated in each graph. ( D ) Allogeneic T cell stimulation. Cell proliferation dye labeled T cells from control donors were incubated for 4 days with MoDCs from patients of the indicated groups. After incubation, cells were harvested, labeled with CD4, CD8 and CD25 and analyzed by flow cytometry. Cell proliferation data shows the frequency of cells within the CD4 + or CD8 + T populations that diluted the proliferation dye, therefore, that proliferated during the experiments. CD25 expression, represented as median fluorescence intensity (MFI) was gated on CD4 T cells. We used one-way ANOVA to compare experimental groups. In all cases, samples from ICC patients were significantly different from the other patients. CC – cervicitis, LG – cells from patients with low grade lesions (or CIN1), HG – cells from patients with high grade lesions (CIN2/3), ICC – cells from patients with invasive cervical carcinoma. ( E ) Pearson correlations between T cell proliferation determined in D and plasma G-CSF concentrations, determined in B; R and p values are indicated in the plots.

Techniques Used: Enzyme-linked Immunosorbent Assay, Immunocytochemistry, Labeling, Concentration Assay, Cell Stimulation, Incubation, Flow Cytometry, Cytometry, Expressing, Fluorescence

11) Product Images from "Epithelial ovarian cancer stem-like cells expressing α-gal epitopes increase the immunogenicity of tumor associated antigens"

Article Title: Epithelial ovarian cancer stem-like cells expressing α-gal epitopes increase the immunogenicity of tumor associated antigens

Journal: BMC Cancer

doi: 10.1186/s12885-015-1973-7

Flow cytometry analysis of levels of CD3 + CD4+ and CD3 + CD8+ T cells in immunized a1,3GT KO mice. Mouse spleen mononuclear cells were isolated by density gradient centrifugation on Ficoll-Paque. And three different colors were used for CD3+, CD4+ and CD8+ membrane markers. Gated CD3 positive events were analyzed for CD4 and CD8 production. The graph of flow cytometry represents data of five experiments with similar results. a The distribution of CD3+CD4+ and CD3+CD8+ T cells in spleen from nonimmunized mice. b The distribution of CD3+CD4+ and CD3+CD8+ T cells in spleen from mice immunized with SKOV3 cells. c The distribution of CD3+CD4+ and CD3+CD8+ T cells in spleen from mice immunized with SKOV3 spheroid cells. d The distribution of CD3+CD4+ and CD3+CD8+ T cells in spleen from mice immunized with SKOV3-gal cells. e The distribution of CD3+CD4+ and CD3+CD8+ T cells in spleen from mice immunized with SKOV3-gal spheroid cells. f The frequency of CD3+CD4+ T-cells. g The frequency of CD3+CD8+ T cells. h The ratio of CD3+CD4+ CD8- cells to CD3+CD4- CD8+ cells (* P
Figure Legend Snippet: Flow cytometry analysis of levels of CD3 + CD4+ and CD3 + CD8+ T cells in immunized a1,3GT KO mice. Mouse spleen mononuclear cells were isolated by density gradient centrifugation on Ficoll-Paque. And three different colors were used for CD3+, CD4+ and CD8+ membrane markers. Gated CD3 positive events were analyzed for CD4 and CD8 production. The graph of flow cytometry represents data of five experiments with similar results. a The distribution of CD3+CD4+ and CD3+CD8+ T cells in spleen from nonimmunized mice. b The distribution of CD3+CD4+ and CD3+CD8+ T cells in spleen from mice immunized with SKOV3 cells. c The distribution of CD3+CD4+ and CD3+CD8+ T cells in spleen from mice immunized with SKOV3 spheroid cells. d The distribution of CD3+CD4+ and CD3+CD8+ T cells in spleen from mice immunized with SKOV3-gal cells. e The distribution of CD3+CD4+ and CD3+CD8+ T cells in spleen from mice immunized with SKOV3-gal spheroid cells. f The frequency of CD3+CD4+ T-cells. g The frequency of CD3+CD8+ T cells. h The ratio of CD3+CD4+ CD8- cells to CD3+CD4- CD8+ cells (* P

Techniques Used: Flow Cytometry, Cytometry, Mouse Assay, Isolation, Gradient Centrifugation

12) Product Images from "A robust intracellular metabolite extraction protocol for human neutrophil metabolic profiling"

Article Title: A robust intracellular metabolite extraction protocol for human neutrophil metabolic profiling

Journal: PLoS ONE

doi: 10.1371/journal.pone.0209270

Optimisation of neutrophil isolation protocols for NMR metabolomics. (A) Spectra (n = 3) shown for neutrophil isolation protocols including (grey spectra) or omitting (red spectra) a wash step immediately after Ficoll-Paque separation. Metabolite loss in the washed samples is indicated by arrows. (B) Spectra (n = 3) show neutrophils incubated with media containing phosphate buffer (red spectra) or HEPES buffer (grey spectra). Multiple contaminating peaks from HEPES are indicated by arrows. (C) Spectra (n = 3) are shown for sample preparation including (grey spectra) or excluding (red spectra) a heat-shock step immediately prior to snap freezing of pellet. Metabolite degradation/turnover was prevented by heat shock as indicated by arrows.
Figure Legend Snippet: Optimisation of neutrophil isolation protocols for NMR metabolomics. (A) Spectra (n = 3) shown for neutrophil isolation protocols including (grey spectra) or omitting (red spectra) a wash step immediately after Ficoll-Paque separation. Metabolite loss in the washed samples is indicated by arrows. (B) Spectra (n = 3) show neutrophils incubated with media containing phosphate buffer (red spectra) or HEPES buffer (grey spectra). Multiple contaminating peaks from HEPES are indicated by arrows. (C) Spectra (n = 3) are shown for sample preparation including (grey spectra) or excluding (red spectra) a heat-shock step immediately prior to snap freezing of pellet. Metabolite degradation/turnover was prevented by heat shock as indicated by arrows.

Techniques Used: Isolation, Nuclear Magnetic Resonance, Incubation, Sample Prep

13) Product Images from "Involvement of Macrophages in the Pathogenesis of Familial Amyloid Polyneuropathy and Efficacy of Human iPS Cell-Derived Macrophages in Its Treatment"

Article Title: Involvement of Macrophages in the Pathogenesis of Familial Amyloid Polyneuropathy and Efficacy of Human iPS Cell-Derived Macrophages in Its Treatment

Journal: PLoS ONE

doi: 10.1371/journal.pone.0163944

Decreased frequency of intracellular TTR in CD14 + monocytes from FAP ATTR V30M patients. CD14 + monocytes were isolated from PBMC of HD ( n = 7) or FAP ATTR V30M patients ( n = 6) using Ficoll-Paque and magnet bead-conjugated anti-human CD14 antibodies. (A, B) Separated CD14 + monocytes were stained with an anti-human TTR antibody. Five visual fields in each stained section were randomly chosen, and the number of TTR-positive CD14 + monocytes counted by three independent observers. The average count number per visual field is shown. The bar graph (A) shows the frequency of TTR + cells in CD14 + monocytes. Photographs (B) show representative data for each group. Scale bars indicate 25 μm. (A, B) The proportion of repeated count TTR + CD14 + monocytes were analyzed using the mixed model, with * p
Figure Legend Snippet: Decreased frequency of intracellular TTR in CD14 + monocytes from FAP ATTR V30M patients. CD14 + monocytes were isolated from PBMC of HD ( n = 7) or FAP ATTR V30M patients ( n = 6) using Ficoll-Paque and magnet bead-conjugated anti-human CD14 antibodies. (A, B) Separated CD14 + monocytes were stained with an anti-human TTR antibody. Five visual fields in each stained section were randomly chosen, and the number of TTR-positive CD14 + monocytes counted by three independent observers. The average count number per visual field is shown. The bar graph (A) shows the frequency of TTR + cells in CD14 + monocytes. Photographs (B) show representative data for each group. Scale bars indicate 25 μm. (A, B) The proportion of repeated count TTR + CD14 + monocytes were analyzed using the mixed model, with * p

Techniques Used: Isolation, Staining

14) Product Images from "Cystatin F Ensures Eosinophil Survival by Regulating Granule Biogenesis"

Article Title: Cystatin F Ensures Eosinophil Survival by Regulating Granule Biogenesis

Journal: Immunity

doi: 10.1016/j.immuni.2016.03.003

CF Stabilizes Granule Integrity and Eosinophil Viability (A) Naive mice received a single dose of BrdU 16 hr before sacrifice assessment of incorporation into eosinophils in the bone marrow. Example flow cytometry plots are shown together with means ± SEM from two independent experiments (n = 4 per group). (B) Bone marrow cells from WT or Cst7 −/− mice were cultured for 4 days in Flt-3L and SCF-1 prior to removal of dead cells by centrifugation over Ficoll-Paque. One million viable cells were seeded into IL-5 medium and eosinophil expansion and survival was monitored by cell counting and flow cytometric identification of Siglec F + cells. Means ± SD of five independent experiments are shown ( ∗ p
Figure Legend Snippet: CF Stabilizes Granule Integrity and Eosinophil Viability (A) Naive mice received a single dose of BrdU 16 hr before sacrifice assessment of incorporation into eosinophils in the bone marrow. Example flow cytometry plots are shown together with means ± SEM from two independent experiments (n = 4 per group). (B) Bone marrow cells from WT or Cst7 −/− mice were cultured for 4 days in Flt-3L and SCF-1 prior to removal of dead cells by centrifugation over Ficoll-Paque. One million viable cells were seeded into IL-5 medium and eosinophil expansion and survival was monitored by cell counting and flow cytometric identification of Siglec F + cells. Means ± SD of five independent experiments are shown ( ∗ p

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Cell Culture, Centrifugation, Cell Counting

15) Product Images from "A robust intracellular metabolite extraction protocol for human neutrophil metabolic profiling"

Article Title: A robust intracellular metabolite extraction protocol for human neutrophil metabolic profiling

Journal: PLoS ONE

doi: 10.1371/journal.pone.0209270

Optimisation of neutrophil isolation protocols for NMR metabolomics. (A) Spectra (n = 3) shown for neutrophil isolation protocols including (grey spectra) or omitting (red spectra) a wash step immediately after Ficoll-Paque separation. Metabolite loss in the washed samples is indicated by arrows. (B) Spectra (n = 3) show neutrophils incubated with media containing phosphate buffer (red spectra) or HEPES buffer (grey spectra). Multiple contaminating peaks from HEPES are indicated by arrows. (C) Spectra (n = 3) are shown for sample preparation including (grey spectra) or excluding (red spectra) a heat-shock step immediately prior to snap freezing of pellet. Metabolite degradation/turnover was prevented by heat shock as indicated by arrows.
Figure Legend Snippet: Optimisation of neutrophil isolation protocols for NMR metabolomics. (A) Spectra (n = 3) shown for neutrophil isolation protocols including (grey spectra) or omitting (red spectra) a wash step immediately after Ficoll-Paque separation. Metabolite loss in the washed samples is indicated by arrows. (B) Spectra (n = 3) show neutrophils incubated with media containing phosphate buffer (red spectra) or HEPES buffer (grey spectra). Multiple contaminating peaks from HEPES are indicated by arrows. (C) Spectra (n = 3) are shown for sample preparation including (grey spectra) or excluding (red spectra) a heat-shock step immediately prior to snap freezing of pellet. Metabolite degradation/turnover was prevented by heat shock as indicated by arrows.

Techniques Used: Isolation, Nuclear Magnetic Resonance, Incubation, Sample Prep

Related Articles

High Performance Liquid Chromatography:

Article Title: A robust intracellular metabolite extraction protocol for human neutrophil metabolic profiling
Article Snippet: .. Materials HetaSep solution was from StemCell (Cambridge, UK); Ficoll-Paque was from GE Healthcare (Little Chalfont, UK); RPMI 1640 media was from Life Technologies (Paisley, UK); phorbol 12-myristate 13-acetate (PMA) and NADP was from Sigma (Gillingham, UK); HPLC grade acetonitrile (Thermofisher, UK), d4 trimethylsilyl propionate (TSP), and NaN3 (Sigma, Gillingham, UK). .. Neutrophil isolation This study was carried out in accordance with the recommendations of the University of Liverpool Committee on Research Ethics.

Centrifugation:

Article Title: Importance of Membrane Fusion Mediated by Human Immunodeficiency Virus Envelope Glycoproteins for Lysis of Primary CD4-Positive T Cells
Article Snippet: .. Whole human peripheral blood mononuclear cells (PBMC) were isolated from fresh blood by Ficoll-Paque (Amersham Pharmacia, Uppsala, Sweden) density centrifugation. ..

Article Title: Glucocorticoid-induced TNF receptor-triggered T cells are key modulators for survival/death of neural stem/progenitor cells induced by ischemic stroke
Article Snippet: .. The mononuclear cells were separated by Ficoll-paque plus (GE Healthcare, Piscataway, NJ, USA) centrifugation, and labeled with antibody cocktails (PerCP-CD3 (BD Biosciences), FITC-CD4 (eBioscience), PE-GITR (BD Biosciences) and APC-CD25 (eBioscience)). .. Rat IgG2a (eBioscience) was used as control isotype staining.

Article Title: Envelope Is a Major Viral Determinant of the Distinct In Vitro Cellular Transformation Tropism of Human T-Cell Leukemia Virus Type 1 (HTLV-1) and HTLV-2
Article Snippet: .. Human PBMCs were isolated from the blood of normal donors by centrifugation over Ficoll-Paque (Amersham Biosciences, Piscataway, NJ) and were cultured in RPMI 1640 medium supplemented with 20% FBS, 2 mM glutamine, and antibiotics. .. In selected experiments, transformed T lymphocytes (10 weeks postcoculture) were treated with 10 U/ml of human interleukin-2 (IL-2) (Roche, Indianapolis, IN) to enhance the short-term growth of cells required for molecular analysis.

Isolation:

Article Title: Importance of Membrane Fusion Mediated by Human Immunodeficiency Virus Envelope Glycoproteins for Lysis of Primary CD4-Positive T Cells
Article Snippet: .. Whole human peripheral blood mononuclear cells (PBMC) were isolated from fresh blood by Ficoll-Paque (Amersham Pharmacia, Uppsala, Sweden) density centrifugation. ..

Article Title: Envelope Is a Major Viral Determinant of the Distinct In Vitro Cellular Transformation Tropism of Human T-Cell Leukemia Virus Type 1 (HTLV-1) and HTLV-2
Article Snippet: .. Human PBMCs were isolated from the blood of normal donors by centrifugation over Ficoll-Paque (Amersham Biosciences, Piscataway, NJ) and were cultured in RPMI 1640 medium supplemented with 20% FBS, 2 mM glutamine, and antibiotics. .. In selected experiments, transformed T lymphocytes (10 weeks postcoculture) were treated with 10 U/ml of human interleukin-2 (IL-2) (Roche, Indianapolis, IN) to enhance the short-term growth of cells required for molecular analysis.

Article Title: Direct measurement of lymphocyte receptor diversity
Article Snippet: .. Lymphocytes were isolated from the resulting suspension of splenocytes or from peripheral blood using Ficoll-paque™ (Amersham Biosciences, Piscataway, NJ) gradient. .. Total RNA was isolated from the lymphocytes using the Qiagen RNeasy Kit™ (Qiagen Inc., Valencia, CA) as per the manufacturer’s instructions.

Article Title: Six‐gene Assay as a new biomarker in the blood of patients with colorectal cancer: establishment and clinical validation
Article Snippet: .. Mononuclear cells from each peripheral blood sample were isolated by density separation with Ficoll‐Paque (GE Healthcare, Little Chalfont, Buckinghamshire, UK). .. RNA was extracted from mononuclear cells using an RNeasy® Plus Micro Kit (Qiagen, Duesseldorf, Germany) according to the manufacturer's instructions.

Cell Culture:

Article Title: Envelope Is a Major Viral Determinant of the Distinct In Vitro Cellular Transformation Tropism of Human T-Cell Leukemia Virus Type 1 (HTLV-1) and HTLV-2
Article Snippet: .. Human PBMCs were isolated from the blood of normal donors by centrifugation over Ficoll-Paque (Amersham Biosciences, Piscataway, NJ) and were cultured in RPMI 1640 medium supplemented with 20% FBS, 2 mM glutamine, and antibiotics. .. In selected experiments, transformed T lymphocytes (10 weeks postcoculture) were treated with 10 U/ml of human interleukin-2 (IL-2) (Roche, Indianapolis, IN) to enhance the short-term growth of cells required for molecular analysis.

Nuclear Magnetic Resonance:

Article Title: A robust intracellular metabolite extraction protocol for human neutrophil metabolic profiling
Article Snippet: .. Supporting information Absence of Ficoll Paque in neutrophil NMR spectra. ..

Labeling:

Article Title: Glucocorticoid-induced TNF receptor-triggered T cells are key modulators for survival/death of neural stem/progenitor cells induced by ischemic stroke
Article Snippet: .. The mononuclear cells were separated by Ficoll-paque plus (GE Healthcare, Piscataway, NJ, USA) centrifugation, and labeled with antibody cocktails (PerCP-CD3 (BD Biosciences), FITC-CD4 (eBioscience), PE-GITR (BD Biosciences) and APC-CD25 (eBioscience)). .. Rat IgG2a (eBioscience) was used as control isotype staining.

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    GE Healthcare ficoll paque plus
    Workflow of sample processing and movement of cells in the FCMC device. (A) Workflow of sample processing and CTC detection. (a) Blood sample collection and enrichment using <t>Ficoll-Paque</t> <t>PLUS.</t> (b) The cell suspension is loaded into the FCMC device. The cells form monolayers in the microchambers and are immunostained by using a syringe pump. (c) All microchambers are scanned using a fluorescent microscope. (d) CTC are detected from the fluorescent images. (B) Movement of cells in the FCMC device. (a) The new CM chip, which is made from polystyrene (PS), is cleaned with UV ozone and coated with BSA. (b) The FCMC device is washed with PBS. (c) A cell suspension (68 uL) that is equivalent to 0.4 mL of blood is loaded into one FCMC device. (d) After “Suction for Trapping”, untrapped cells move into downstream microchambers and settle down on the bottom of these microchambers. The blue arrows indicate flow. (e) After “Suction for Monolayer”, overlapped cells are discharged from microchambers and move to the outside of microchambers or to the inside of downstream microchambers. (f) After repetitions of (d) and (e), all cells are trapped in a monolayer. (g), (h), (i) Immunostaining solutions are loaded, incubated and washed.
    Ficoll Paque Plus, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 95/100, based on 3410 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    GE Healthcare ficoll paque premium
    Experimental design and purity isolated cells. (A) Monocytes isolated from the blood of healthy donors were differentiated into macrophages by 7-day culture with M-CSF, and the resulting macrophages were cultured for 18 hours with polarizing factors or medium and then for three days with or without the probiotic VSL#3. (B–D) PBMC and monocyte samples were taken on day 0 after <t>Ficoll-Paque</t> centrifugation and magnetic bead purification, respectively, while the macrophage sample was taken from adhered cells on day 7. Cells were stained with anti-CD3-PerCP and anti-CD14-FITC antibodies and analyzed on a FACSAria flow cytometer. Representative graphs are shown.
    Ficoll Paque Premium, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    GE Healthcare ficoll paque plus medium
    BHLHE40-knockdown (KD) reduced lung colonization of tumor cells inoculated into the circulation via tail veins. a Less tumor cells were detected in blood of NSG mice inoculated via tail vein injection with LM BHLHE40-KD than mice inoculated with empty vector (EV) control LM EV cells. Tumor cells in whole blood collected by cardiac puncture at indicated times after tail vein injection were isolated using the <t>Ficoll-Paque</t> <t>PLUS</t> medium and counted under fluorescent microscope. * p
    Ficoll Paque Plus Medium, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 89/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Workflow of sample processing and movement of cells in the FCMC device. (A) Workflow of sample processing and CTC detection. (a) Blood sample collection and enrichment using Ficoll-Paque PLUS. (b) The cell suspension is loaded into the FCMC device. The cells form monolayers in the microchambers and are immunostained by using a syringe pump. (c) All microchambers are scanned using a fluorescent microscope. (d) CTC are detected from the fluorescent images. (B) Movement of cells in the FCMC device. (a) The new CM chip, which is made from polystyrene (PS), is cleaned with UV ozone and coated with BSA. (b) The FCMC device is washed with PBS. (c) A cell suspension (68 uL) that is equivalent to 0.4 mL of blood is loaded into one FCMC device. (d) After “Suction for Trapping”, untrapped cells move into downstream microchambers and settle down on the bottom of these microchambers. The blue arrows indicate flow. (e) After “Suction for Monolayer”, overlapped cells are discharged from microchambers and move to the outside of microchambers or to the inside of downstream microchambers. (f) After repetitions of (d) and (e), all cells are trapped in a monolayer. (g), (h), (i) Immunostaining solutions are loaded, incubated and washed.

    Journal: EBioMedicine

    Article Title: Prognostic Impact of Circulating Tumor Cell Detected Using a Novel Fluidic Cell Microarray Chip System in Patients with Breast Cancer

    doi: 10.1016/j.ebiom.2016.07.027

    Figure Lengend Snippet: Workflow of sample processing and movement of cells in the FCMC device. (A) Workflow of sample processing and CTC detection. (a) Blood sample collection and enrichment using Ficoll-Paque PLUS. (b) The cell suspension is loaded into the FCMC device. The cells form monolayers in the microchambers and are immunostained by using a syringe pump. (c) All microchambers are scanned using a fluorescent microscope. (d) CTC are detected from the fluorescent images. (B) Movement of cells in the FCMC device. (a) The new CM chip, which is made from polystyrene (PS), is cleaned with UV ozone and coated with BSA. (b) The FCMC device is washed with PBS. (c) A cell suspension (68 uL) that is equivalent to 0.4 mL of blood is loaded into one FCMC device. (d) After “Suction for Trapping”, untrapped cells move into downstream microchambers and settle down on the bottom of these microchambers. The blue arrows indicate flow. (e) After “Suction for Monolayer”, overlapped cells are discharged from microchambers and move to the outside of microchambers or to the inside of downstream microchambers. (f) After repetitions of (d) and (e), all cells are trapped in a monolayer. (g), (h), (i) Immunostaining solutions are loaded, incubated and washed.

    Article Snippet: Two milliliters of blood sample were processed using Ficoll-Paque PLUS (GE Healthcare, Little Chalfont, UK).

    Techniques: Microscopy, Chromatin Immunoprecipitation, Flow Cytometry, Immunostaining, Incubation

    Figure 1. Western blot showing histone H3 and H4 acetylation in leukocytes separated by (A) Ficoll-Paque PLUS, or (B) red cell lysis. 20µg of protein was loaded in each lane of 15% SDS-PAGE gels. Rabbit anti-acetyl histone H3 detected

    Journal: Epigenetics

    Article Title: Methods for the analysis of histone H3 and H4 acetylation in blood

    doi: 10.4161/epi.20983

    Figure Lengend Snippet: Figure 1. Western blot showing histone H3 and H4 acetylation in leukocytes separated by (A) Ficoll-Paque PLUS, or (B) red cell lysis. 20µg of protein was loaded in each lane of 15% SDS-PAGE gels. Rabbit anti-acetyl histone H3 detected

    Article Snippet: An equal volume of PBS was added to 2 mL blood, mixed and overlaid onto 3 mL of Ficoll-Paque PLUS (GE Life Sciences).

    Techniques: Western Blot, Lysis, SDS Page

    Experimental design and purity isolated cells. (A) Monocytes isolated from the blood of healthy donors were differentiated into macrophages by 7-day culture with M-CSF, and the resulting macrophages were cultured for 18 hours with polarizing factors or medium and then for three days with or without the probiotic VSL#3. (B–D) PBMC and monocyte samples were taken on day 0 after Ficoll-Paque centrifugation and magnetic bead purification, respectively, while the macrophage sample was taken from adhered cells on day 7. Cells were stained with anti-CD3-PerCP and anti-CD14-FITC antibodies and analyzed on a FACSAria flow cytometer. Representative graphs are shown.

    Journal: Journal of clinical & cellular immunology

    Article Title: The Probiotic Mixture VSL#3 Alters the Morphology and Secretion Profile of Both Polarized and Unpolarized Human Macrophages in a Polarization-Dependent Manner

    doi: 10.4172/2155-9899.1000227

    Figure Lengend Snippet: Experimental design and purity isolated cells. (A) Monocytes isolated from the blood of healthy donors were differentiated into macrophages by 7-day culture with M-CSF, and the resulting macrophages were cultured for 18 hours with polarizing factors or medium and then for three days with or without the probiotic VSL#3. (B–D) PBMC and monocyte samples were taken on day 0 after Ficoll-Paque centrifugation and magnetic bead purification, respectively, while the macrophage sample was taken from adhered cells on day 7. Cells were stained with anti-CD3-PerCP and anti-CD14-FITC antibodies and analyzed on a FACSAria flow cytometer. Representative graphs are shown.

    Article Snippet: Peripheral blood mononuclear cells (PBMCs) from blood diluted ~1:3.5 in (PBS + 2 mM EDTA) were obtained by density gradient centrifugation with Ficoll-Paque Premium (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and pooled in equal parts in order to obtain an equal representation from each volunteer [ , ].

    Techniques: Isolation, Cell Culture, Centrifugation, Purification, Staining, Flow Cytometry, Cytometry

    BHLHE40-knockdown (KD) reduced lung colonization of tumor cells inoculated into the circulation via tail veins. a Less tumor cells were detected in blood of NSG mice inoculated via tail vein injection with LM BHLHE40-KD than mice inoculated with empty vector (EV) control LM EV cells. Tumor cells in whole blood collected by cardiac puncture at indicated times after tail vein injection were isolated using the Ficoll-Paque PLUS medium and counted under fluorescent microscope. * p

    Journal: Breast Cancer Research : BCR

    Article Title: BHLHE40 confers a pro-survival and pro-metastatic phenotype to breast cancer cells by modulating HBEGF secretion

    doi: 10.1186/s13058-018-1046-3

    Figure Lengend Snippet: BHLHE40-knockdown (KD) reduced lung colonization of tumor cells inoculated into the circulation via tail veins. a Less tumor cells were detected in blood of NSG mice inoculated via tail vein injection with LM BHLHE40-KD than mice inoculated with empty vector (EV) control LM EV cells. Tumor cells in whole blood collected by cardiac puncture at indicated times after tail vein injection were isolated using the Ficoll-Paque PLUS medium and counted under fluorescent microscope. * p

    Article Snippet: Circulating tumor cells in whole blood collected by cardiac puncture were isolated using the Ficoll-Paque PLUS medium (GE Healthcare Life Sciences, Piscataway, NJ, USA) and counted under fluorescent microscope.

    Techniques: Mouse Assay, Injection, Plasmid Preparation, Isolation, Microscopy