Journal: PLoS ONE

Article Title: Analysis of cell surface and intranuclear markers on non-stimulated human PBMC using mass cytometry

doi: 10.1371/journal.pone.0194593

Figure Lengend Snippet: Effects of different buffers on the detection of rare populations. Cells from healthy donors were labelled with antibodies targeting both cell surface and intranuclear antigens. Different permeabilization conditions are compared to the cell surface staining only” (CS) condition: ICSb (BD cytofix/cytoperm buffer), INSb 1 (eBioscience permeabilization buffer), INSb 2 (Maxpar NASB) and INSb 3 (Methanol/PFA). This data compare the frequency of various CD45+ populations between the CS condition and the different permeabilization conditions. a.) Frequencies of rare CD4+ T cell populations: primed T cells (CXCR5+ CCR7+) and Treg cells (CD25 hi CD127 low ). b.) Frequencies of rare B cell populations such as transitional B cells (CD24 hi CD38 hi ) and un-switched memory B cells (CD19+IgD+CD27+). c.) Statistics showing the comparison of the frequency of rare T and B cell populations within the different experimental conditions. The concentrations of antibodies used were: CXCR5 (0.04 mg/ml), CCR7 (0.5 mg/ml), CD25 (0.5 mg/ml), CD127 (0.5 mg/ml), CD24 (0.3 mg/ml), CD38 (0.3 mg/ml), IgD (0.25 mg/ml) and CD27 (0.1 mg/ml). Statistics was performed using one-way ANOVA with Bonferroni’s multiple test correction (*p

Article Snippet: Fixation and permeabilization using BD cytofix/cytoperm buffer (BD, France, Catalog # 554715) Cells were incubated at RT for 1 hour with BD Cytofix/Cytoperm buffer, then washed twice with BD wash/perm solution diluted 10X in distilled water.

Techniques: Staining

Journal: PLoS ONE

Article Title: Analysis of cell surface and intranuclear markers on non-stimulated human PBMC using mass cytometry

doi: 10.1371/journal.pone.0194593

Figure Lengend Snippet: Detection of intranuclear markers using the adapted BD cytofix/cytoperm protocol. Cells from healthy donors were incubated with antibodies targeting cell surface antigens and then split into 5 for the following conditions: the “surface staining only” conditions (CS), and fixation and permeabilization using either BD cytofix/cytoperm buffer (ICSb), eBioscience fixation and permeabilization buffer (INSb 1), Maxpar NASB (INSb 2) and PFA/methanol (INSb 3). Next cells were labelled with a mix of antibodies targeting intranuclear markers. Data compare the frequencies of Treg cells (CD25hiFoxP3+), Tfh cells (CD4+BCL6+), Th17 cells (CD4+RoryT+) and CD8+Tbet+ cells between the various permeabilization conditions. The concentrations of antibodies used are as follow: FoxP3 (0.3 mg/ml), BCL6 (0.8 mg/ml), RoryT (0.6 mg/ml) and Tbet (0.3 mg/ml). Statistics was performed using one-way ANOVA with Bonferroni’s multiple test correction (*p

Article Snippet: Fixation and permeabilization using BD cytofix/cytoperm buffer (BD, France, Catalog # 554715) Cells were incubated at RT for 1 hour with BD Cytofix/Cytoperm buffer, then washed twice with BD wash/perm solution diluted 10X in distilled water.

Techniques: Incubation, Staining

Journal: PLoS ONE

Article Title: Analysis of cell surface and intranuclear markers on non-stimulated human PBMC using mass cytometry

doi: 10.1371/journal.pone.0194593

Figure Lengend Snippet: Visualization of the effects of different buffers on the intensity of cell surface markers. Cells from healthy donors were labeled with antibodies targeting both cell surface antigens and intranuclear antigens. Data show the distribution of CD45+ live cells on viSNE plots with the “cell surface staining” only condition (CS) and the different permeabilization conditions: ICSb (BD cytofix/cytoperm buffer), INSb 1 (eBioscience permeabilization buffer), INSb 2 (Maxpar NASB) and INSb 3 (Methanol/PFA). The intensity of CD19+, CD16+, CD56+, CD14+ and HLADR+ events are shown. The concentrations of antibodies used for the detection of these markers are as follows: CD19 (0.5 mg/ml), CD16 (0.2 mg/ml), CD56 (0.1 mg/ml), CD14 (0.3 mg/ml) and HLADR (0.3 mg/ml). viSNE was performed using 1000 iterations, with a perplexity of 30 and theta = 0.3. The data shown are representative of an independent experiment and represent median with interquartile. Experiments were performed 3 times independently. Different healthy individuals were used for each independent experiment.

Article Snippet: Fixation and permeabilization using BD cytofix/cytoperm buffer (BD, France, Catalog # 554715) Cells were incubated at RT for 1 hour with BD Cytofix/Cytoperm buffer, then washed twice with BD wash/perm solution diluted 10X in distilled water.

Techniques: Labeling, Staining

Journal: PLoS ONE

Article Title: Analysis of cell surface and intranuclear markers on non-stimulated human PBMC using mass cytometry

doi: 10.1371/journal.pone.0194593

Figure Lengend Snippet: Partial loss of the signal intensity of CD4 and CD127 after barcoding. Cells were thawed as described above and used for the following purposes: a) assessment of the effects of barcoding on the expression of surface markers. Three experimental conditions were performed: no barcode/no permeabilization, surface staining before barcoding and finally barcoding before surface staining. Data show histogram overlays of CD45, CD3, CD4, CD127 and CD25 for the different conditions. The concentrations of antibodies used are as follow: CD45 (0.5 mg/ml), CD3 (0.08 mg/ml), CD4 (0.25 mg/ml), CD127 (0.5 mg/ml) and CD25 (0.5 mg/ml). b) Assessment of the cause of the lower signal intensity of CD4 and CD127 when PBMC are barcoded before cell surface staining. 3 conditions were evaluated: no barcode/no permeabilization, permeabilization only/no barcoding and permeabilization followed by barcoding. Data show histogram overlays of CD45, CD3, CD4, CD127 and CD25 for the different conditions. The data shown are representative of an independent experiment. Experiments were performed 3 times independently. PBMC from different individuals were used for each independent experiment.

Article Snippet: Fixation and permeabilization using BD cytofix/cytoperm buffer (BD, France, Catalog # 554715) Cells were incubated at RT for 1 hour with BD Cytofix/Cytoperm buffer, then washed twice with BD wash/perm solution diluted 10X in distilled water.

Techniques: Expressing, Staining

Journal: PLoS ONE

Article Title: Analysis of cell surface and intranuclear markers on non-stimulated human PBMC using mass cytometry

doi: 10.1371/journal.pone.0194593

Figure Lengend Snippet: Effects of different buffers on the detection of cell surface markers. Cells from healthy donors were labelled with antibodies targeting both cell surface and intranuclear antigens. Different permeabilization conditions are compared to the cell surface staining only” (CS) condition: ICSb (BD cytofix/cytoperm buffer), INSb 1 (eBioscience permeabilization buffer), INSb 2 (Maxpar NASB) and INSb 3 (Methanol/PFA). Here, we show the effects of different permeabilization conditions on the frequency of various cell surface markers. a.) Histograms showing the frequency and distribution of CD45+, CD19+, CD3+, CD16+, CD56+, CD14+ and HLADR+ events in the CS condition. The concentrations of antibodies used were: CD45 (0.5 mg/ml), CD19 (0.5 mg/ml), CD3 (0.08 mg/ml), CD16 (0.2 mg/ml), CD56 (0.1 mg/ml), CD14 (0.3 mg/ml) and HLADR (0.3 mg/ml). b.) Comparison of the frequency of CD45+, CD19+, CD3+, CD16+, CD56+, CD14+ and HLADR+ events between the CS condition and the different permeabilization conditions. Statistics was performed using one-way ANOVA with Bonferroni’s multiple test correction (*p

Article Snippet: Fixation and permeabilization using BD cytofix/cytoperm buffer (BD, France, Catalog # 554715) Cells were incubated at RT for 1 hour with BD Cytofix/Cytoperm buffer, then washed twice with BD wash/perm solution diluted 10X in distilled water.

Techniques: Staining