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Becton Dickinson bd facsaria iii cell sorter
RNA-seq of stained and sorted cell populations A : Bioanalyzer profiles of total RNA derived from unprocessed cells (harvested directly from trypsinised cells) compared to cells that have undergone glyoxal fixation, permeabilisation with 100% methanol and indirect immunofluorescence for CCNB1. 0.1-5 ng of total RNA was separated on total RNA pico chips on Agilent 2100 Bioanalyzer. B : Scatter plots comparing the normalised read count for each annotated gene in GRCh38 in poly(A)+ RNA-seq libraries derived from unprocessed and from glyoxal fixed, permeabilised and stained cells. Two biological replicates were sequenced for each condition and averaged, read counts were normalised to the total number of reads in each library. Data is shown for both COLO205 and MCF-7 cells. C : Flow cytometry density plots for MCF-7 cells labelled with anti-CCNB1 primary antibody and donkey Alexa Fluor-488 conjugated secondary antibody and sorted using a BD <t>FACSAria</t> <t>III</t> sorter. Intact cells (shown within the elliptical gate) were distinguished from off-scale events and cell debris using forward scatter (FSC) and side scatter (SSC) measurements (panel 1). Doublets were excluded from the gated cells by a 2-step gating strategy with pulse height (H) plotted against area (A) for FSC parameter followed by SSC-H versus SSC-A plot (panel 3). Fluorescence thresholds for isolation of CCNB1 positive and negative cell fractions are shown in panel 4. Gates were set using the negative control and the CCNB1 positive and negative sorting gates were moved apart from each other to maximise purity when sorted. D : Fluorescence histogram of DAPI intensities for sorted CCNB1 negative and positive populations E : Scatter plots comparing the normalised read count for each annotated gene in GRCh38 in poly(A)+ RNA-seq libraries derived from two biological replicates each of CCNB1 positive (left) and negative (right) MCF-7 cells. Note the particularly tight correlation after sorting towards the antigen of interest. E : Hierarchical clustering analysis of the 161 significantly different expressed genes identified DEseq2 analysis (p
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1) Product Images from "Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry"

Article Title: Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry

Journal: bioRxiv

doi: 10.1101/2020.10.05.326082

RNA-seq of stained and sorted cell populations A : Bioanalyzer profiles of total RNA derived from unprocessed cells (harvested directly from trypsinised cells) compared to cells that have undergone glyoxal fixation, permeabilisation with 100% methanol and indirect immunofluorescence for CCNB1. 0.1-5 ng of total RNA was separated on total RNA pico chips on Agilent 2100 Bioanalyzer. B : Scatter plots comparing the normalised read count for each annotated gene in GRCh38 in poly(A)+ RNA-seq libraries derived from unprocessed and from glyoxal fixed, permeabilised and stained cells. Two biological replicates were sequenced for each condition and averaged, read counts were normalised to the total number of reads in each library. Data is shown for both COLO205 and MCF-7 cells. C : Flow cytometry density plots for MCF-7 cells labelled with anti-CCNB1 primary antibody and donkey Alexa Fluor-488 conjugated secondary antibody and sorted using a BD FACSAria III sorter. Intact cells (shown within the elliptical gate) were distinguished from off-scale events and cell debris using forward scatter (FSC) and side scatter (SSC) measurements (panel 1). Doublets were excluded from the gated cells by a 2-step gating strategy with pulse height (H) plotted against area (A) for FSC parameter followed by SSC-H versus SSC-A plot (panel 3). Fluorescence thresholds for isolation of CCNB1 positive and negative cell fractions are shown in panel 4. Gates were set using the negative control and the CCNB1 positive and negative sorting gates were moved apart from each other to maximise purity when sorted. D : Fluorescence histogram of DAPI intensities for sorted CCNB1 negative and positive populations E : Scatter plots comparing the normalised read count for each annotated gene in GRCh38 in poly(A)+ RNA-seq libraries derived from two biological replicates each of CCNB1 positive (left) and negative (right) MCF-7 cells. Note the particularly tight correlation after sorting towards the antigen of interest. E : Hierarchical clustering analysis of the 161 significantly different expressed genes identified DEseq2 analysis (p
Figure Legend Snippet: RNA-seq of stained and sorted cell populations A : Bioanalyzer profiles of total RNA derived from unprocessed cells (harvested directly from trypsinised cells) compared to cells that have undergone glyoxal fixation, permeabilisation with 100% methanol and indirect immunofluorescence for CCNB1. 0.1-5 ng of total RNA was separated on total RNA pico chips on Agilent 2100 Bioanalyzer. B : Scatter plots comparing the normalised read count for each annotated gene in GRCh38 in poly(A)+ RNA-seq libraries derived from unprocessed and from glyoxal fixed, permeabilised and stained cells. Two biological replicates were sequenced for each condition and averaged, read counts were normalised to the total number of reads in each library. Data is shown for both COLO205 and MCF-7 cells. C : Flow cytometry density plots for MCF-7 cells labelled with anti-CCNB1 primary antibody and donkey Alexa Fluor-488 conjugated secondary antibody and sorted using a BD FACSAria III sorter. Intact cells (shown within the elliptical gate) were distinguished from off-scale events and cell debris using forward scatter (FSC) and side scatter (SSC) measurements (panel 1). Doublets were excluded from the gated cells by a 2-step gating strategy with pulse height (H) plotted against area (A) for FSC parameter followed by SSC-H versus SSC-A plot (panel 3). Fluorescence thresholds for isolation of CCNB1 positive and negative cell fractions are shown in panel 4. Gates were set using the negative control and the CCNB1 positive and negative sorting gates were moved apart from each other to maximise purity when sorted. D : Fluorescence histogram of DAPI intensities for sorted CCNB1 negative and positive populations E : Scatter plots comparing the normalised read count for each annotated gene in GRCh38 in poly(A)+ RNA-seq libraries derived from two biological replicates each of CCNB1 positive (left) and negative (right) MCF-7 cells. Note the particularly tight correlation after sorting towards the antigen of interest. E : Hierarchical clustering analysis of the 161 significantly different expressed genes identified DEseq2 analysis (p

Techniques Used: RNA Sequencing Assay, Staining, Derivative Assay, Immunofluorescence, Flow Cytometry, Fluorescence, Isolation, Negative Control

2) Product Images from "Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry"

Article Title: Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry

Journal: PLoS ONE

doi: 10.1371/journal.pone.0240769

RNA-seq of stained and sorted cell populations. A : Bioanalyzer profiles of total RNA derived from unprocessed cells (harvested directly from trypsinised cells) compared to cells that have undergone glyoxal fixation, permeabilisation with 100% methanol and indirect immunofluorescence for CCNB1. 0.1–5 ng of total RNA was separated on total RNA pico chips on Agilent 2100 Bioanalyzer. B : Scatter plots comparing the normalised read count for each annotated gene in GRCh38 in poly(A)+ RNA-seq libraries derived from unprocessed and from glyoxal fixed, permeabilised and stained cells. Two biological replicates were sequenced for each condition and averaged, read counts were normalised to the total number of reads in each library. Data is shown for both COLO205 and MCF-7 cells. C : Flow cytometry density plots for MCF-7 cells labelled with anti-CCNB1 primary antibody and donkey Alexa Fluor-488 conjugated secondary antibody and sorted using a BD FACSAria III sorter. Intact cells (shown within the elliptical gate) were distinguished from off-scale events and cell debris using forward scatter (FSC) and side scatter (SSC) measurements (panel 1). Doublets were excluded from the gated cells by a 2-step gating strategy with pulse height (H) plotted against area (A) for FSC parameter followed by SSC-H versus SSC-A plot (panel 3). Fluorescence thresholds for isolation of CCNB1 positive and negative cell fractions are shown in panel 4. Gates were set using the negative control and the CCNB1 positive and negative sorting gates were moved apart from each other to maximise purity when sorted. D : Fluorescence histogram of DAPI intensities for sorted CCNB1 negative and positive populations. E : Scatter plots comparing the normalised read count for each annotated gene in GRCh38 in poly(A)+ RNA-seq libraries derived from two biological replicates each of CCNB1 positive (left) and negative (right) MCF-7 cells. Note the particularly tight correlation after sorting towards the antigen of interest. E : Hierarchical clustering analysis of the 161 significantly different expressed genes identified DEseq2 analysis (p
Figure Legend Snippet: RNA-seq of stained and sorted cell populations. A : Bioanalyzer profiles of total RNA derived from unprocessed cells (harvested directly from trypsinised cells) compared to cells that have undergone glyoxal fixation, permeabilisation with 100% methanol and indirect immunofluorescence for CCNB1. 0.1–5 ng of total RNA was separated on total RNA pico chips on Agilent 2100 Bioanalyzer. B : Scatter plots comparing the normalised read count for each annotated gene in GRCh38 in poly(A)+ RNA-seq libraries derived from unprocessed and from glyoxal fixed, permeabilised and stained cells. Two biological replicates were sequenced for each condition and averaged, read counts were normalised to the total number of reads in each library. Data is shown for both COLO205 and MCF-7 cells. C : Flow cytometry density plots for MCF-7 cells labelled with anti-CCNB1 primary antibody and donkey Alexa Fluor-488 conjugated secondary antibody and sorted using a BD FACSAria III sorter. Intact cells (shown within the elliptical gate) were distinguished from off-scale events and cell debris using forward scatter (FSC) and side scatter (SSC) measurements (panel 1). Doublets were excluded from the gated cells by a 2-step gating strategy with pulse height (H) plotted against area (A) for FSC parameter followed by SSC-H versus SSC-A plot (panel 3). Fluorescence thresholds for isolation of CCNB1 positive and negative cell fractions are shown in panel 4. Gates were set using the negative control and the CCNB1 positive and negative sorting gates were moved apart from each other to maximise purity when sorted. D : Fluorescence histogram of DAPI intensities for sorted CCNB1 negative and positive populations. E : Scatter plots comparing the normalised read count for each annotated gene in GRCh38 in poly(A)+ RNA-seq libraries derived from two biological replicates each of CCNB1 positive (left) and negative (right) MCF-7 cells. Note the particularly tight correlation after sorting towards the antigen of interest. E : Hierarchical clustering analysis of the 161 significantly different expressed genes identified DEseq2 analysis (p

Techniques Used: RNA Sequencing Assay, Staining, Derivative Assay, Immunofluorescence, Flow Cytometry, Fluorescence, Isolation, Negative Control

Related Articles

Staining:

Article Title: ACME dissociation: a versatile cell fixation-dissociation method for single-cell transcriptomics
Article Snippet: Staining concentrations require optimization and ultimately depend on FACS/Cytometer adjustments and cell concentration. .. Cells were stained in the dark, on ice, for 30–45 min, and visualized using a CytoFlex S Flow Cytometer (Beckman Coulter) or sorted using a BD FACS Aria III (BD Biosciences) Cell Sorter. .. To avoid RNAse contamination during cell sorting, the FACS was thoroughly decontaminated with bleach and pre-cooled before sorting, keeping injection and collection chambers at 4 °C during the process.

Flow Cytometry:

Article Title: ACME dissociation: a versatile cell fixation-dissociation method for single-cell transcriptomics
Article Snippet: Staining concentrations require optimization and ultimately depend on FACS/Cytometer adjustments and cell concentration. .. Cells were stained in the dark, on ice, for 30–45 min, and visualized using a CytoFlex S Flow Cytometer (Beckman Coulter) or sorted using a BD FACS Aria III (BD Biosciences) Cell Sorter. .. To avoid RNAse contamination during cell sorting, the FACS was thoroughly decontaminated with bleach and pre-cooled before sorting, keeping injection and collection chambers at 4 °C during the process.

Article Title: CD200–CD200R immune checkpoint engagement regulates ILC2 effector function and ameliorates lung inflammation in asthma
Article Snippet: Data were analyzed with FlowJo software (TreeStar) version 10. .. ILC2 sorting and ex vivo stimulation All cells were purified by flow cytometry using BD FACS ARIA III (BD biosciences, San Jose, CA) with a purity of > 95%. .. The isolated ILC2s were cultured (at least 5 × 103 cells/well) in 96-well round-bottom plates with Gibco™ Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Waltham, MA) that was supplemented with 10% fetal bovine serum (FBS), 2% antibiotics (penicillin and streptomycin), and 0.05 mM b-mercaptoethanol.

FACS:

Article Title: ACME dissociation: a versatile cell fixation-dissociation method for single-cell transcriptomics
Article Snippet: Staining concentrations require optimization and ultimately depend on FACS/Cytometer adjustments and cell concentration. .. Cells were stained in the dark, on ice, for 30–45 min, and visualized using a CytoFlex S Flow Cytometer (Beckman Coulter) or sorted using a BD FACS Aria III (BD Biosciences) Cell Sorter. .. To avoid RNAse contamination during cell sorting, the FACS was thoroughly decontaminated with bleach and pre-cooled before sorting, keeping injection and collection chambers at 4 °C during the process.

Article Title: DJ-1 depletion slows down immunoaging in T-cell compartments
Article Snippet: Adoptive transfer of naïve CD8+ T cells CD90.2+ cells from spleen and pLNs of DJ-1-/- mice and DJ-1+/+ littermates aged 8-12 weeks or ∼55 weeks were magnetically isolated and first enriched by using anti-mouse CD90.2 microbeads (Miltenyi Biotec, 130-049-101, also refer to ). .. Naïve CD8+ T cells (CD3+ CD8+ CD44low CD62Lhi ) were then sorted by FACS sorting (BD FACSAria™ III sorter (The sorting antibody information was already provided in ). .. 2.5E5 purified naïve CD8+ T cells in 100 ul of PBS (Lonza, BE17-516F) were injected intravenously into 8-12 week-old Rag-1 -/- mice.

Article Title: CD200–CD200R immune checkpoint engagement regulates ILC2 effector function and ameliorates lung inflammation in asthma
Article Snippet: Data were analyzed with FlowJo software (TreeStar) version 10. .. ILC2 sorting and ex vivo stimulation All cells were purified by flow cytometry using BD FACS ARIA III (BD biosciences, San Jose, CA) with a purity of > 95%. .. The isolated ILC2s were cultured (at least 5 × 103 cells/well) in 96-well round-bottom plates with Gibco™ Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Waltham, MA) that was supplemented with 10% fetal bovine serum (FBS), 2% antibiotics (penicillin and streptomycin), and 0.05 mM b-mercaptoethanol.

Article Title: GATA transcription factors, SOX17 and TFAP2C, drive the human germ-cell specification program
Article Snippet: The images of the aggregates were taken under an M205C stereo microscope (Leica Microsystems) equipped with a DP72 CCD camera and DP2-BSW software (Olympus). .. FACSFor analysis of the cellular contents of the aggregates, the aggregates were collected on the designated days of induction, washed once in PBS, and dissociated with 0.25% Trypsin–EDTA for 10–15 min at 37°C with gentle pipetting every 5 min. Trypsin was neutralized with a 5× volume of 10% FBS in DMEM, and the resuspended cells were processed with FACS Aria III system (BD Biosciences) and analyzed with FACS Diva software. .. The method for selecting CRISPR-mediated knockout clones is described in the section “Generation of knockout lines.”.

Article Title: Zeb1-Hdac2-eNOS circuitry identifies early cardiovascular precursors in naive mouse embryonic stem cells
Article Snippet: Sorting setup and appropriate gating were established each time using mESC cultured in DM in the absence of DAF-2DA. .. To minimize cell death and maximize cell recovery sorting was performed by using FACS ARIA III from BD (Beckton Dickinson). ..

Article Title: The Function of FK506-Binding Protein 13 in Protein Quality Control Protects Plasma Cells from Endoplasmic Reticulum Stress-Associated Apoptosis
Article Snippet: .. Enzyme-Linked Immunosorbent Assays (ELISA)Sixteen hours after transfection of J558 cells with MigR1, MigR1-myc-FKBP13, pGFP-V-RS, or pGFP-V-RS-shFKBP13, GFP-positive cells were sorted using a FACS Aria III (BD Biosciences) and cultured in DMEM with 10% horse serum. .. After 48 h, supernatants were collected and assayed by sandwich ELISA to measure secreted IgA.

Article Title: The transcription factor EGR2 is the molecular linchpin connecting STAT6 activation to the late, stable epigenomic program of alternative macrophage polarization
Article Snippet: For intracellular detection of RELMα, Zenon Alexa fluor 350 rabbit IgG labeling kit (Thermo Fischer Scientific) was used to add fluorescent label to RELMα (Peprotech) antibody, BD Cytofix Cytoperm fixation/permeabilization solution kit (BD Biosciences) was used for fixation/permeabilization and staining according to the manufacturer's recommendations. .. Samples were acquired on a BD FACS Aria III (BD Biosciences) cell sorter using BD FACSDiva 6.0 software (BD Biosciences). .. Data analysis was performed using FlowJo v10 software (Becton Dickinson and Company).

Ex Vivo:

Article Title: CD200–CD200R immune checkpoint engagement regulates ILC2 effector function and ameliorates lung inflammation in asthma
Article Snippet: Data were analyzed with FlowJo software (TreeStar) version 10. .. ILC2 sorting and ex vivo stimulation All cells were purified by flow cytometry using BD FACS ARIA III (BD biosciences, San Jose, CA) with a purity of > 95%. .. The isolated ILC2s were cultured (at least 5 × 103 cells/well) in 96-well round-bottom plates with Gibco™ Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Waltham, MA) that was supplemented with 10% fetal bovine serum (FBS), 2% antibiotics (penicillin and streptomycin), and 0.05 mM b-mercaptoethanol.

Purification:

Article Title: CD200–CD200R immune checkpoint engagement regulates ILC2 effector function and ameliorates lung inflammation in asthma
Article Snippet: Data were analyzed with FlowJo software (TreeStar) version 10. .. ILC2 sorting and ex vivo stimulation All cells were purified by flow cytometry using BD FACS ARIA III (BD biosciences, San Jose, CA) with a purity of > 95%. .. The isolated ILC2s were cultured (at least 5 × 103 cells/well) in 96-well round-bottom plates with Gibco™ Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Waltham, MA) that was supplemented with 10% fetal bovine serum (FBS), 2% antibiotics (penicillin and streptomycin), and 0.05 mM b-mercaptoethanol.

Software:

Article Title: GATA transcription factors, SOX17 and TFAP2C, drive the human germ-cell specification program
Article Snippet: The images of the aggregates were taken under an M205C stereo microscope (Leica Microsystems) equipped with a DP72 CCD camera and DP2-BSW software (Olympus). .. FACSFor analysis of the cellular contents of the aggregates, the aggregates were collected on the designated days of induction, washed once in PBS, and dissociated with 0.25% Trypsin–EDTA for 10–15 min at 37°C with gentle pipetting every 5 min. Trypsin was neutralized with a 5× volume of 10% FBS in DMEM, and the resuspended cells were processed with FACS Aria III system (BD Biosciences) and analyzed with FACS Diva software. .. The method for selecting CRISPR-mediated knockout clones is described in the section “Generation of knockout lines.”.

Article Title: The transcription factor EGR2 is the molecular linchpin connecting STAT6 activation to the late, stable epigenomic program of alternative macrophage polarization
Article Snippet: For intracellular detection of RELMα, Zenon Alexa fluor 350 rabbit IgG labeling kit (Thermo Fischer Scientific) was used to add fluorescent label to RELMα (Peprotech) antibody, BD Cytofix Cytoperm fixation/permeabilization solution kit (BD Biosciences) was used for fixation/permeabilization and staining according to the manufacturer's recommendations. .. Samples were acquired on a BD FACS Aria III (BD Biosciences) cell sorter using BD FACSDiva 6.0 software (BD Biosciences). .. Data analysis was performed using FlowJo v10 software (Becton Dickinson and Company).

Enzyme-linked Immunosorbent Assay:

Article Title: The Function of FK506-Binding Protein 13 in Protein Quality Control Protects Plasma Cells from Endoplasmic Reticulum Stress-Associated Apoptosis
Article Snippet: .. Enzyme-Linked Immunosorbent Assays (ELISA)Sixteen hours after transfection of J558 cells with MigR1, MigR1-myc-FKBP13, pGFP-V-RS, or pGFP-V-RS-shFKBP13, GFP-positive cells were sorted using a FACS Aria III (BD Biosciences) and cultured in DMEM with 10% horse serum. .. After 48 h, supernatants were collected and assayed by sandwich ELISA to measure secreted IgA.

Transfection:

Article Title: The Function of FK506-Binding Protein 13 in Protein Quality Control Protects Plasma Cells from Endoplasmic Reticulum Stress-Associated Apoptosis
Article Snippet: .. Enzyme-Linked Immunosorbent Assays (ELISA)Sixteen hours after transfection of J558 cells with MigR1, MigR1-myc-FKBP13, pGFP-V-RS, or pGFP-V-RS-shFKBP13, GFP-positive cells were sorted using a FACS Aria III (BD Biosciences) and cultured in DMEM with 10% horse serum. .. After 48 h, supernatants were collected and assayed by sandwich ELISA to measure secreted IgA.

Cell Culture:

Article Title: The Function of FK506-Binding Protein 13 in Protein Quality Control Protects Plasma Cells from Endoplasmic Reticulum Stress-Associated Apoptosis
Article Snippet: .. Enzyme-Linked Immunosorbent Assays (ELISA)Sixteen hours after transfection of J558 cells with MigR1, MigR1-myc-FKBP13, pGFP-V-RS, or pGFP-V-RS-shFKBP13, GFP-positive cells were sorted using a FACS Aria III (BD Biosciences) and cultured in DMEM with 10% horse serum. .. After 48 h, supernatants were collected and assayed by sandwich ELISA to measure secreted IgA.

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    Becton Dickinson facsaria iii cell sorter
    Repression of activation-induced cytidine deaminase (AID) induction in germinal center B cells (GCBs) from Pten fl/fl Cγ1 Cre/+ mice. (A) Flow cytometry sorting of in vitro induced IgM-BCR expressing splenic B cells from Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ mice. Pure splenic B cells from control and KO mice (three mice for each group) were stimulated with LPS plus interleukin-4 (IL-4) for 4 days before sorting. (B) The expression levels of PTEN, p-AKT, p-FoxO1/3a, and AID were determined in Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells (left). Reduced PTEN, AID expression and enhanced p-AKT and p-FoxO1/3a expression were observed in Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells. The in vitro induced IgM-BCR expressing B cells were isolated by the <t>FACSAria</t> <t>III</t> Cell Sorter as described in (A) . Statistical comparison of protein expression level was also shown in right. Ratio values of different proteins from Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells were normalized to that of the Pten +/+ Cγ1 Cre/+ IgM-BCR expressing B cells. Data were given from one representative of at least three independent experiments. * p
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    Becton Dickinson facs sorted
    NPC treatment alters the gene expression signature of CNS-infiltrating inflammatory MCs in EAE. ( A ) Cohorts of 4–7 MOG 35–55 -immunized C57BL/6 mice intrathecally treated with either PBS or NPCs at the peak of the disease (2–4 days after clinical onset). At 7 days after transplantation, CNS tissues were pooled and CNS-infiltrating MCs were <t>FACS-sorted</t> according to the phenotype CD45 hi Ly6G – <t>CD11b</t> + Ly6C hi MHC-II + . Sorting strategy used for 3 independent FACS sorting experiments is shown. ( B ) Next-generation sequencing was performed on RNA extracted from sorted cells of 3 independent experiments and respective CNS harvests. Six hundred ten genes that are significantly altered in 3 different statistical tests to a minimum significance threshold of P ≤ 0.01 are shown in the heatmap. ( C ) Volcano plot showing the fold change and significance of genes in MCs from PBS- versus NPC-treated EAE mice. ( D ). Black and gray nodes represent enriched pathways with sizes corresponding to FDR-adjusted enrichment P value ( P ≤ 0.05). Red dots represent upregulated genes and blue dots downregulated genes, whereas the dot size indicates significance ( P ≤ 0.01).
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    Becton Dickinson bd facsaria iii cell sorter
    RNA-seq of stained and sorted cell populations A : Bioanalyzer profiles of total RNA derived from unprocessed cells (harvested directly from trypsinised cells) compared to cells that have undergone glyoxal fixation, permeabilisation with 100% methanol and indirect immunofluorescence for CCNB1. 0.1-5 ng of total RNA was separated on total RNA pico chips on Agilent 2100 Bioanalyzer. B : Scatter plots comparing the normalised read count for each annotated gene in GRCh38 in poly(A)+ RNA-seq libraries derived from unprocessed and from glyoxal fixed, permeabilised and stained cells. Two biological replicates were sequenced for each condition and averaged, read counts were normalised to the total number of reads in each library. Data is shown for both COLO205 and MCF-7 cells. C : Flow cytometry density plots for MCF-7 cells labelled with anti-CCNB1 primary antibody and donkey Alexa Fluor-488 conjugated secondary antibody and sorted using a BD <t>FACSAria</t> <t>III</t> sorter. Intact cells (shown within the elliptical gate) were distinguished from off-scale events and cell debris using forward scatter (FSC) and side scatter (SSC) measurements (panel 1). Doublets were excluded from the gated cells by a 2-step gating strategy with pulse height (H) plotted against area (A) for FSC parameter followed by SSC-H versus SSC-A plot (panel 3). Fluorescence thresholds for isolation of CCNB1 positive and negative cell fractions are shown in panel 4. Gates were set using the negative control and the CCNB1 positive and negative sorting gates were moved apart from each other to maximise purity when sorted. D : Fluorescence histogram of DAPI intensities for sorted CCNB1 negative and positive populations E : Scatter plots comparing the normalised read count for each annotated gene in GRCh38 in poly(A)+ RNA-seq libraries derived from two biological replicates each of CCNB1 positive (left) and negative (right) MCF-7 cells. Note the particularly tight correlation after sorting towards the antigen of interest. E : Hierarchical clustering analysis of the 161 significantly different expressed genes identified DEseq2 analysis (p
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    Becton Dickinson facs aria iii flow sorter
    CCL3 and CCL4 are upregulated and secreted by activated CTLs and mediate homotypic recruitment. ( A, B ) Quantification of chemokine mRNA levels by qRT-PCR in OT1 ( A ) or gBT1 ( B ) CTLs exposed to H-2K b /SIINFEKL cognate beads, or cognate SIINFEKL- or SSIEFARL-pulsed EL4 respectively, or non-cognate unpulsed EL4 tumour cells, as indicated. Data is normalised to universal mouse cDNA (positive control). Data points from four independent experiments; bars indicate mean. ND: not detected. ( C ) Quantification of absolute chemokine concentration detected in non-cognate and cognate OT1 supernatants by ELISA (for CCL1 and CCL9) or CBA (for CCL3, CCL4, CCL5, CXCL10, IFN-γ, and TNF-α). Data points from four independent experiments; red lines indicate mean. ND: not detected. ( D ) Transmigration of OT1 CTLs towards basal medium containing indicated concentrations of recombinant chemokine. Data normalised to basal medium (dashed line). Data points from <t>three</t> independent experiments; bars indicate mean. Error bars represent standard deviation. p- values are displayed when means are significantly different compared to the 1 ng/ml condition. ( E ) Transmigration of OT1 CTLs towards chemokine-containing basal medium in the presence of corresponding neutralising antibodies or isotype (IgG) control antibody. Data points from four independent experiments; bars indicate mean. Error bars represent standard deviation. ( F ) Transmigration of CTLs towards cognate supernatant in the presence of neutralising antibodies or IgG isotype control. Data normalised to basal medium (dashed line). Data points from at least three independent experiments; bars indicate mean. Error bars represent standard deviation. p-values are displayed when means are significantly different to both the negative control and the IgG isotype control conditions. ( G ) FMI of OT1 CTLs adjacent to tumouroids containing non-cognate or cognate tumour cells with pre-embedded tumour-reactive CTLs in the presence of chemokine-neutralising antibodies as indicated. ( H ) Time course of CCL3 and CCL4 concentrations in supernatants from CTLs sorted by <t>FACS</t> from 4 hr conjugations with cognate tumour cells (0 hr: completion of conjugation). Solid line: cumulative concentration. Dotted line: differential concentration per hour (difference of cumulative concentrations at consecutive timepoints divided by number of hours in-between). Error bars and shaded areas: range. ( I ) FMI of OT1 CTLs adjacent to masses containing CTLs only (no tumour cells) sorted from conjugations with cognate or non-cognate tumour cells. Comparisons of transmigration indices in ( C ) to ( E ) were performed using ANOVA and Tukey’s multiple comparisons tests. In ( G ) and ( I ), box-whiskers indicate medians and the interquartile range (IQR) with outliers outside whiskers. ns: p > 0.05, p-values from two-tailed Wilcoxon signed rank test compared to a theoretical median of 0. Source data file for Figure 4—figure supplement 1 .
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    Repression of activation-induced cytidine deaminase (AID) induction in germinal center B cells (GCBs) from Pten fl/fl Cγ1 Cre/+ mice. (A) Flow cytometry sorting of in vitro induced IgM-BCR expressing splenic B cells from Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ mice. Pure splenic B cells from control and KO mice (three mice for each group) were stimulated with LPS plus interleukin-4 (IL-4) for 4 days before sorting. (B) The expression levels of PTEN, p-AKT, p-FoxO1/3a, and AID were determined in Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells (left). Reduced PTEN, AID expression and enhanced p-AKT and p-FoxO1/3a expression were observed in Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells. The in vitro induced IgM-BCR expressing B cells were isolated by the FACSAria III Cell Sorter as described in (A) . Statistical comparison of protein expression level was also shown in right. Ratio values of different proteins from Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells were normalized to that of the Pten +/+ Cγ1 Cre/+ IgM-BCR expressing B cells. Data were given from one representative of at least three independent experiments. * p

    Journal: Frontiers in Immunology

    Article Title: PTEN-Regulated AID Transcription in Germinal Center B Cells Is Essential for the Class-Switch Recombination and IgG Antibody Responses

    doi: 10.3389/fimmu.2018.00371

    Figure Lengend Snippet: Repression of activation-induced cytidine deaminase (AID) induction in germinal center B cells (GCBs) from Pten fl/fl Cγ1 Cre/+ mice. (A) Flow cytometry sorting of in vitro induced IgM-BCR expressing splenic B cells from Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ mice. Pure splenic B cells from control and KO mice (three mice for each group) were stimulated with LPS plus interleukin-4 (IL-4) for 4 days before sorting. (B) The expression levels of PTEN, p-AKT, p-FoxO1/3a, and AID were determined in Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells (left). Reduced PTEN, AID expression and enhanced p-AKT and p-FoxO1/3a expression were observed in Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells. The in vitro induced IgM-BCR expressing B cells were isolated by the FACSAria III Cell Sorter as described in (A) . Statistical comparison of protein expression level was also shown in right. Ratio values of different proteins from Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells were normalized to that of the Pten +/+ Cγ1 Cre/+ IgM-BCR expressing B cells. Data were given from one representative of at least three independent experiments. * p

    Article Snippet: Pre-incubated IgM-BCR expressing B cells were sorted by using FACSAria III Cell Sorter (BD).

    Techniques: Activation Assay, Mouse Assay, Flow Cytometry, Cytometry, In Vitro, Expressing, Isolation

    NPC treatment alters the gene expression signature of CNS-infiltrating inflammatory MCs in EAE. ( A ) Cohorts of 4–7 MOG 35–55 -immunized C57BL/6 mice intrathecally treated with either PBS or NPCs at the peak of the disease (2–4 days after clinical onset). At 7 days after transplantation, CNS tissues were pooled and CNS-infiltrating MCs were FACS-sorted according to the phenotype CD45 hi Ly6G – CD11b + Ly6C hi MHC-II + . Sorting strategy used for 3 independent FACS sorting experiments is shown. ( B ) Next-generation sequencing was performed on RNA extracted from sorted cells of 3 independent experiments and respective CNS harvests. Six hundred ten genes that are significantly altered in 3 different statistical tests to a minimum significance threshold of P ≤ 0.01 are shown in the heatmap. ( C ) Volcano plot showing the fold change and significance of genes in MCs from PBS- versus NPC-treated EAE mice. ( D ). Black and gray nodes represent enriched pathways with sizes corresponding to FDR-adjusted enrichment P value ( P ≤ 0.05). Red dots represent upregulated genes and blue dots downregulated genes, whereas the dot size indicates significance ( P ≤ 0.01).

    Journal: The Journal of Clinical Investigation

    Article Title: Neural precursor cell–secreted TGF- β2 redirects inflammatory monocyte-derived cells in CNS autoimmunity

    doi: 10.1172/JCI92387

    Figure Lengend Snippet: NPC treatment alters the gene expression signature of CNS-infiltrating inflammatory MCs in EAE. ( A ) Cohorts of 4–7 MOG 35–55 -immunized C57BL/6 mice intrathecally treated with either PBS or NPCs at the peak of the disease (2–4 days after clinical onset). At 7 days after transplantation, CNS tissues were pooled and CNS-infiltrating MCs were FACS-sorted according to the phenotype CD45 hi Ly6G – CD11b + Ly6C hi MHC-II + . Sorting strategy used for 3 independent FACS sorting experiments is shown. ( B ) Next-generation sequencing was performed on RNA extracted from sorted cells of 3 independent experiments and respective CNS harvests. Six hundred ten genes that are significantly altered in 3 different statistical tests to a minimum significance threshold of P ≤ 0.01 are shown in the heatmap. ( C ) Volcano plot showing the fold change and significance of genes in MCs from PBS- versus NPC-treated EAE mice. ( D ). Black and gray nodes represent enriched pathways with sizes corresponding to FDR-adjusted enrichment P value ( P ≤ 0.05). Red dots represent upregulated genes and blue dots downregulated genes, whereas the dot size indicates significance ( P ≤ 0.01).

    Article Snippet: Seven days after transplantation, CD45hi CD11b+ Ly6Chi Ly6G– MHC-II+ MCs were FACS-sorted (BD FACSAria III) from hindbrain and spinal cord as described above.

    Techniques: Expressing, Mouse Assay, Transplantation Assay, FACS, Next-Generation Sequencing

    RNA-seq of stained and sorted cell populations A : Bioanalyzer profiles of total RNA derived from unprocessed cells (harvested directly from trypsinised cells) compared to cells that have undergone glyoxal fixation, permeabilisation with 100% methanol and indirect immunofluorescence for CCNB1. 0.1-5 ng of total RNA was separated on total RNA pico chips on Agilent 2100 Bioanalyzer. B : Scatter plots comparing the normalised read count for each annotated gene in GRCh38 in poly(A)+ RNA-seq libraries derived from unprocessed and from glyoxal fixed, permeabilised and stained cells. Two biological replicates were sequenced for each condition and averaged, read counts were normalised to the total number of reads in each library. Data is shown for both COLO205 and MCF-7 cells. C : Flow cytometry density plots for MCF-7 cells labelled with anti-CCNB1 primary antibody and donkey Alexa Fluor-488 conjugated secondary antibody and sorted using a BD FACSAria III sorter. Intact cells (shown within the elliptical gate) were distinguished from off-scale events and cell debris using forward scatter (FSC) and side scatter (SSC) measurements (panel 1). Doublets were excluded from the gated cells by a 2-step gating strategy with pulse height (H) plotted against area (A) for FSC parameter followed by SSC-H versus SSC-A plot (panel 3). Fluorescence thresholds for isolation of CCNB1 positive and negative cell fractions are shown in panel 4. Gates were set using the negative control and the CCNB1 positive and negative sorting gates were moved apart from each other to maximise purity when sorted. D : Fluorescence histogram of DAPI intensities for sorted CCNB1 negative and positive populations E : Scatter plots comparing the normalised read count for each annotated gene in GRCh38 in poly(A)+ RNA-seq libraries derived from two biological replicates each of CCNB1 positive (left) and negative (right) MCF-7 cells. Note the particularly tight correlation after sorting towards the antigen of interest. E : Hierarchical clustering analysis of the 161 significantly different expressed genes identified DEseq2 analysis (p

    Journal: bioRxiv

    Article Title: Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry

    doi: 10.1101/2020.10.05.326082

    Figure Lengend Snippet: RNA-seq of stained and sorted cell populations A : Bioanalyzer profiles of total RNA derived from unprocessed cells (harvested directly from trypsinised cells) compared to cells that have undergone glyoxal fixation, permeabilisation with 100% methanol and indirect immunofluorescence for CCNB1. 0.1-5 ng of total RNA was separated on total RNA pico chips on Agilent 2100 Bioanalyzer. B : Scatter plots comparing the normalised read count for each annotated gene in GRCh38 in poly(A)+ RNA-seq libraries derived from unprocessed and from glyoxal fixed, permeabilised and stained cells. Two biological replicates were sequenced for each condition and averaged, read counts were normalised to the total number of reads in each library. Data is shown for both COLO205 and MCF-7 cells. C : Flow cytometry density plots for MCF-7 cells labelled with anti-CCNB1 primary antibody and donkey Alexa Fluor-488 conjugated secondary antibody and sorted using a BD FACSAria III sorter. Intact cells (shown within the elliptical gate) were distinguished from off-scale events and cell debris using forward scatter (FSC) and side scatter (SSC) measurements (panel 1). Doublets were excluded from the gated cells by a 2-step gating strategy with pulse height (H) plotted against area (A) for FSC parameter followed by SSC-H versus SSC-A plot (panel 3). Fluorescence thresholds for isolation of CCNB1 positive and negative cell fractions are shown in panel 4. Gates were set using the negative control and the CCNB1 positive and negative sorting gates were moved apart from each other to maximise purity when sorted. D : Fluorescence histogram of DAPI intensities for sorted CCNB1 negative and positive populations E : Scatter plots comparing the normalised read count for each annotated gene in GRCh38 in poly(A)+ RNA-seq libraries derived from two biological replicates each of CCNB1 positive (left) and negative (right) MCF-7 cells. Note the particularly tight correlation after sorting towards the antigen of interest. E : Hierarchical clustering analysis of the 161 significantly different expressed genes identified DEseq2 analysis (p

    Article Snippet: Cells were sorted using a 100 μm nozzle (at 20PSI) on a BD FACSARIA III cell sorter (BD Biosciences, UK) with cooling of the sample and collection chambers enabled.

    Techniques: RNA Sequencing Assay, Staining, Derivative Assay, Immunofluorescence, Flow Cytometry, Fluorescence, Isolation, Negative Control

    CCL3 and CCL4 are upregulated and secreted by activated CTLs and mediate homotypic recruitment. ( A, B ) Quantification of chemokine mRNA levels by qRT-PCR in OT1 ( A ) or gBT1 ( B ) CTLs exposed to H-2K b /SIINFEKL cognate beads, or cognate SIINFEKL- or SSIEFARL-pulsed EL4 respectively, or non-cognate unpulsed EL4 tumour cells, as indicated. Data is normalised to universal mouse cDNA (positive control). Data points from four independent experiments; bars indicate mean. ND: not detected. ( C ) Quantification of absolute chemokine concentration detected in non-cognate and cognate OT1 supernatants by ELISA (for CCL1 and CCL9) or CBA (for CCL3, CCL4, CCL5, CXCL10, IFN-γ, and TNF-α). Data points from four independent experiments; red lines indicate mean. ND: not detected. ( D ) Transmigration of OT1 CTLs towards basal medium containing indicated concentrations of recombinant chemokine. Data normalised to basal medium (dashed line). Data points from three independent experiments; bars indicate mean. Error bars represent standard deviation. p- values are displayed when means are significantly different compared to the 1 ng/ml condition. ( E ) Transmigration of OT1 CTLs towards chemokine-containing basal medium in the presence of corresponding neutralising antibodies or isotype (IgG) control antibody. Data points from four independent experiments; bars indicate mean. Error bars represent standard deviation. ( F ) Transmigration of CTLs towards cognate supernatant in the presence of neutralising antibodies or IgG isotype control. Data normalised to basal medium (dashed line). Data points from at least three independent experiments; bars indicate mean. Error bars represent standard deviation. p-values are displayed when means are significantly different to both the negative control and the IgG isotype control conditions. ( G ) FMI of OT1 CTLs adjacent to tumouroids containing non-cognate or cognate tumour cells with pre-embedded tumour-reactive CTLs in the presence of chemokine-neutralising antibodies as indicated. ( H ) Time course of CCL3 and CCL4 concentrations in supernatants from CTLs sorted by FACS from 4 hr conjugations with cognate tumour cells (0 hr: completion of conjugation). Solid line: cumulative concentration. Dotted line: differential concentration per hour (difference of cumulative concentrations at consecutive timepoints divided by number of hours in-between). Error bars and shaded areas: range. ( I ) FMI of OT1 CTLs adjacent to masses containing CTLs only (no tumour cells) sorted from conjugations with cognate or non-cognate tumour cells. Comparisons of transmigration indices in ( C ) to ( E ) were performed using ANOVA and Tukey’s multiple comparisons tests. In ( G ) and ( I ), box-whiskers indicate medians and the interquartile range (IQR) with outliers outside whiskers. ns: p > 0.05, p-values from two-tailed Wilcoxon signed rank test compared to a theoretical median of 0. Source data file for Figure 4—figure supplement 1 .

    Journal: eLife

    Article Title: Cytotoxic T cells swarm by homotypic chemokine signalling

    doi: 10.7554/eLife.56554

    Figure Lengend Snippet: CCL3 and CCL4 are upregulated and secreted by activated CTLs and mediate homotypic recruitment. ( A, B ) Quantification of chemokine mRNA levels by qRT-PCR in OT1 ( A ) or gBT1 ( B ) CTLs exposed to H-2K b /SIINFEKL cognate beads, or cognate SIINFEKL- or SSIEFARL-pulsed EL4 respectively, or non-cognate unpulsed EL4 tumour cells, as indicated. Data is normalised to universal mouse cDNA (positive control). Data points from four independent experiments; bars indicate mean. ND: not detected. ( C ) Quantification of absolute chemokine concentration detected in non-cognate and cognate OT1 supernatants by ELISA (for CCL1 and CCL9) or CBA (for CCL3, CCL4, CCL5, CXCL10, IFN-γ, and TNF-α). Data points from four independent experiments; red lines indicate mean. ND: not detected. ( D ) Transmigration of OT1 CTLs towards basal medium containing indicated concentrations of recombinant chemokine. Data normalised to basal medium (dashed line). Data points from three independent experiments; bars indicate mean. Error bars represent standard deviation. p- values are displayed when means are significantly different compared to the 1 ng/ml condition. ( E ) Transmigration of OT1 CTLs towards chemokine-containing basal medium in the presence of corresponding neutralising antibodies or isotype (IgG) control antibody. Data points from four independent experiments; bars indicate mean. Error bars represent standard deviation. ( F ) Transmigration of CTLs towards cognate supernatant in the presence of neutralising antibodies or IgG isotype control. Data normalised to basal medium (dashed line). Data points from at least three independent experiments; bars indicate mean. Error bars represent standard deviation. p-values are displayed when means are significantly different to both the negative control and the IgG isotype control conditions. ( G ) FMI of OT1 CTLs adjacent to tumouroids containing non-cognate or cognate tumour cells with pre-embedded tumour-reactive CTLs in the presence of chemokine-neutralising antibodies as indicated. ( H ) Time course of CCL3 and CCL4 concentrations in supernatants from CTLs sorted by FACS from 4 hr conjugations with cognate tumour cells (0 hr: completion of conjugation). Solid line: cumulative concentration. Dotted line: differential concentration per hour (difference of cumulative concentrations at consecutive timepoints divided by number of hours in-between). Error bars and shaded areas: range. ( I ) FMI of OT1 CTLs adjacent to masses containing CTLs only (no tumour cells) sorted from conjugations with cognate or non-cognate tumour cells. Comparisons of transmigration indices in ( C ) to ( E ) were performed using ANOVA and Tukey’s multiple comparisons tests. In ( G ) and ( I ), box-whiskers indicate medians and the interquartile range (IQR) with outliers outside whiskers. ns: p > 0.05, p-values from two-tailed Wilcoxon signed rank test compared to a theoretical median of 0. Source data file for Figure 4—figure supplement 1 .

    Article Snippet: Cells were then washed and sorted on the FACS-Aria III flow sorter (BD Biosciences; Franklin Lakes, NJ, USA) based on CD8 and CD44 surface expression detected by antibody staining with 1 µg/ml anti-CD8a-Pacific Blue (clone 53–6.7; BD Biosciences) and anti-CD44-APC (clone IM7; BD Biosciences).

    Techniques: Quantitative RT-PCR, Positive Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Crocin Bleaching Assay, Transmigration Assay, Recombinant, Standard Deviation, Negative Control, FACS, Conjugation Assay, Two Tailed Test