BD FACSCelesta

Overview

The BD FACSCelesta™ flow cytometer can rapidly and accurately analyze many kinds of cells, including lymphoid tissue (thymus, spleen and lymph nodes), digested solid tissues and blood.

Using panels of directly conjugated fluorescent antibodies to recognize specific cell surface and intracellular epitopes, multicolor flow cytometric analysis allows researchers to interrogate specific target protein levels expressed by individual cells in various phases of development and differentiation. The multiparametric, single-cell focus of multicolor flow cytometry is perfectly suited to further immunological discovery of protein and gene expression and cell signaling. BD's solutions for cell identification, transcription factor expression, and cytokine secretion and measurement reflect a commitment to high quality and consistency, which are needed for advanced research.

Illuminate Rare Cells and Low-Density Antigens

Rare cells, or cells that have few surface receptors of a marker of interest, can be difficult to detect using conventional reagents. Bright reagents are essential in resolving these dim cells from others in a sample.

BD Horizon Brilliant™ polymer conjugates can endow previously dim cells with much brighter fluorescence signals than traditional organic fluorescent dyes or even phycobiliproteins such as PE or APC. Optimization for these bright dyes enables the BD FACSCelesta to identify cell populations with a broader range of receptor density than was previously possible.


2-Laser BV Configuration

Ten-color T-cell analysis on the BD FACSCelesta BV configuration
Ten-color T-cell analysis on the BD FACSCelesta BV configuration
This T-cell panel demonstrates the sensitivity and resolution of the BD FACSCelesta flow cytometer, even in detecting rare subpopulations using a 2-laser system. After normal human whole blood was washed and lysed, BD Horizon Brilliant™ and traditional dyes were used to identify rare T-cell and Treg subpopulations. A. Cells were gated to select the CD3+ T cells. B. CD3+ lymphocytes were gated to show the CD4+ helper T cells and CD8+ cytotoxic T cells. C. Gated on the CD4+ T cells, surface markers were used to identify CD25+CD127- Tregs. D. Gated on Tregs, surface markers were used to identify memory and naive Treg subsets (CD194, CD45RA). E. CD4+ helper T cells were analyzed for memory T-cell subsets using CD45RA and CD197. Memory subsets were further defined using CD27 and CD194 surface markers. F. CD8+ cytotoxic T cells were analyzed for memory T-cell subsets using CD45RA and CD197. Memory subsets were further defined using CD28 and CD194 surface markers.

3-Laser BVR Configuration

Six-color Treg analysis on the BD FACSCelesta BVR configuration
Six-color Treg analysis on the BD FACSCelesta BVR configuration
Normal human whole blood was stained, lysed and washed to analyze regulatory T cells (Tregs). Cells were gated and lymphocytes were identified using light scatter. Lymphocytes were further gated on CD3+CD4+ helper T cells and finally on CD25+CD127- Tregs to identify naïve (CD45RO-CD194-) and memory (CD45RO+CD194+) Tregs. (The absence of CD127 is a proxy for the presence of the classic intracellular Treg marker FoxP3.) BD Horizon Brilliant dyes were used to stain four of the six markers, allowing easy resolution of rare Treg subpopulations.
Ten-color T-cell analysis on the BD FACSCelesta BVR configuration
Ten-color T-cell analysis on the BD FACSCelesta BVR configuration
This T-cell panel demonstrates the sensitivity and resolution of the BD FACSCelesta flow cytometer, even in detecting rare subpopulations using a 2-laser system. After normal human whole blood was washed and lysed, BD Horizon Brilliant and traditional dyes were used to identify rare T-cell and Treg subpopulations. A. Cells were gated to select the CD3+ T cells. B. CD3+ lymphocytes were gated to show the CD4+ helper T cells and CD8+ cytotoxic T cells. C. Gated on the CD4+ T cells, surface markers were used to identify CD25+CD127- Tregs. D. Gated on Tregs, surface markers were used to identify memory and naive Treg subserts (CD194, CD45RA). E. CD4+ helper T cells were analyzed for memory T-cell subsets using CD45RO and CD197 (left) and effector memory subsets were further defined with surface markers for CD27 and CD194 (right). F. CD8+ cytotoxic T cells were analyzed for effector and memory subsets using CD45RO and CD197 (left) and effector/effector memory subsets were further defined using CD27 and CD28 surface markers. Similar analysis was completed for CD8+ cytotoxic T cells (right).
Three-color minimal spectral overlap human Treg panel on the BD FACSCelesta BVR configuration
Three-color minimal spectral overlap human Treg panel on the BD FACSCelesta BVR configuration
Human whole blood cells were stained with fluorescent antibodies to Treg markers and acquired and analyzed on the BD FACSCelesta BVR configuration. Lymphocytes were identified based on light scatter properties and CD4 expression, measured using FITC (excited by the blue laser). Tregs were further defined based on CD25 expression using BV421 (violet laser), and CD127 expression using Alexa Fluor® 647 (red laser). Results: Tregs (CD4+CD25highCD127low-) were clearly distinguished, and comparison of compensated (middle plot) and not compensated (right plot) data showed that the plots are virtually identical. The compensation matrix shows minimal spectral overlap using these three fluorochromes together.

Resolution of T-cell markers on the BD FACSCelesta BVR configuration
Resolution of T-cell markers on the BD FACSCelesta BVR configuration
Human whole blood was stained, lysed and fixed prior to analysis on the BD FACSCelesta BVR configuration. Four panel-appropriate fluorochromes were paired with each of three markers varying in antigen density. Results: T cells were first identified based on light scatter properties of lymphocytes and CD3 expression (not shown). Cells expressing high-, medium- and low-antigen density markers (CD4, CD27 and CD132), when paired with each fluorochrome, were clearly resolved from negative or unstained cells. Gates were drawn based on CD3 single-stained controls.
Deep immunophenotyping of mouse dendritic cell subsets
Deep immunophenotyping of mouse dendritic cell subsets
Results of a ten-color immunophenotypic characterization of the three main subsets of mouse DCs (myeloid, lymphoid and plasmacytoid) in the mouse spleen are shown. BALB/c mouse spleen was enzymatically digested, stained with a cocktail of antibodies (including BD OptiBuild™ versions of Sirpα BV650 and Clec12A BV786) and analyzed on a BD FACSCelesta BVR laser configuration. Cells were initially gated on CD3, CD19 and 7-AAD negative cells (not shown). A–D. Gating strategy: I-A/I-EhighCD11chigh conventional dendritic cells (DCs) were further discriminated into CD11b+ mDCs and CD8+ IDCs. I-A/I-ElowCD11clow B220+Gr1+ cells were recognized as pDCs. Results: Differential expression of the additional markers CD4, CD172a (Sirpα) and CD371 (Clec12A) was further analyzed within the mDC (E, F, G), IDC (H, I, J) and pDC (K, L, M) subsets, respectively. Gates were drawn based on fluorescence minus one (FMO) controls.

3-Laser BVYG Configuration

Eight-color NK and Treg analysis on the BD FACSCelesta BVYG configuration
Eight-color NK and Treg analysis on the BD FACSCelesta BVYG configuration
This panel demonstrates the sensitivity and resolution of the BD FACSCelesta flow cytometer configured with the 561-nm yellow-green laser in detecting rare cell subpopulations. After normal human whole blood was washed and lysed, BD Horizon Brilliant and traditional dyes were used to identify rare NK-cell and Treg subpopulations. A. Cells were gated to select the CD3+ or CD3- lymphocytes. B. CD3- lymphocytes were analyzed to show the three subsets of NK cells based on CD16 and CD56 expression. C. Gated on the CD56dimCD16+ NK-cell population, surface markers were used to identify CD57 and CD8 NK-cell subpopulations. D. Gated on CD3+ T cells, CD8- T cells were identified. E. Using surface markers, Tregs were identified from the CD8-CD4+ helper T cells based on CD25 expression. F. Gated on Tregs, Treg activated and resting subsets can be resolved based on HLA-DR expression.

3-Laser BVUV Configuration

Six-color Treg analysis on the BD FACSCelesta BVUV configuration
Six-color Treg analysis on the BD FACSCelesta BVUV configuration
Normal human whole blood was washed, lysed and stained to analyze Tregs and CD4+ helper T-cell subsets. Cells were gated and lymphocytes were identified using light scatter. Lymphocytes were further gated on CD3+CD4+ helper T cells and analyzed for rare T-cell subsets using CD45RO and CD194 surface markers or identification of Tregs (CD25+CD127-). Within the Treg subset, naïve (CD45RO-CD194-) and memory (CD45RO+CD194+) Tregs were identified.
Twelve-color T-cell analysis on the BD FACSCelesta BVUV configuration
Twelve-color T-cell analysis on the BD FACSCelesta BVUV configuration
This T-cell panel demonstrates the sensitivity and resolution of the BD FACSCelesta, even in detecting rare subpopulations. After normal human whole blood was stained, lysed and washed, BD Horizon Brilliant dyes were used to stain nine of the 12 markers, allowing easy resolution of rare T-cell and Treg subpopulations. A. Cells were gated to select the CD3+ T cells. B. CD3+ lymphocytes were gated to show the CD4+ helper T cells and CD8+ cytotoxic T cells. C. Gated on the CD4+ T cells, surface markers were used to identify CD25+CD127- Tregs. (The absence of CD127 is a proxy for the presence of the classic intracellular Treg marker FoxP3.) D. CD4+ helper T cells were analyzed for memory T-cell subsets using CD45RO, CD197 and CD27. Additional surface markers were used to distinguish CD127, CD45RA and CD95 expression levels. E. CD8+ cytotoxic T cells were analyzed for memory T-cell subsets using CD45RO, CD197, CD127 and CD28. F. HLA-DR and CD45RO expression levels were plotted for the Treg population.
Four-color minimal spectral overlap human Treg panel on the BD FACSCelesta BVUV configuration
Four-color minimal spectral overlap human Treg panel on the BD FACSCelesta BVUV configuration
Human whole blood cells were stained with fluorescent antibodies to Treg markers and acquired and analyzed on the BD FACSCelesta BVUV configuration. CD4 and CD25 expression were measured using PerCP-Cy™5.5 and BD Horizon Brilliant™ Blue 515 (BB515), respectively (both excited by the blue laser); CD127 expression using BV421 (violet laser); and CD3 expression using BUV395 (UV laser). Results: Tregs (CD3+CD4+CD25highCD127low/–) were clearly distinguished. The not compensated (shown) and compensated (not shown) data were virtually identical, and the compensation matrix shows minimal spectral overlap using these four fluorochromes together.

Resolution of B-cell markers on the BD FACSCelesta BVUV configuration
Resolution of B-cell markers on the BD FACSCelesta BVUV configuration
Peripheral blood mononuclear cells were isolated and stained prior to analysis on the BD FACSCelesta BVUV configuration. Four panel-appropriate fluorochromes were paired with each of three markers varying in antigen density. Results: Lymphocytes were identified based on light scatter properties. For the analysis of CD38, B cells were further defined based on CD19 expression (not shown). Cells expressing high-, medium- and low-antigen density markers (CD20, CD19 and CD38) were clearly resolved from negative cells. Within the CD19+ cell population, distinct subsets of cells expressing variable levels of CD38, from low to high, were discriminated (right column). Gates were drawn based on unstained (for CD20 and CD19) or CD19 single-stained (for CD38) cells.
Intracellular cytokine response in TLR ligand-activated human dendritic cell subsets
Intracellullar cytokine response in TLR ligand-activated human dendritic cell subsets
Six-color immunophenotypic and functional characterization of the two main subsets of human DCs (plasmacytoid and myeloid) are shown in these plots. Peripheral blood from a normal human donor was treated with lipopolysaccharide (LPS, α TLR4 ligand), Imiquimod (R837, α TLR7 ligand), or Resiquimod (R848, α TLR7/8 ligand) or left unstimulated for four hours in the presence of BD GolgiPlug™ protein transport inhibitor. Samples were stained with a cocktail of antibodies for detection of surface markers, lysed, fixed and permeabilized using BD Cytofix/Cytoperm™ buffers prior to staining with intracellular antibodies for detection of cytokines TNF-α and IFN-α. Cells were analyzed on the BD FACSCelesta BVUV laser configuration. Results: A. Cells were first gated based on light scatter, and then on HLA-DR expression and absence of lineage markers (HLA-DR+CD3CD19CD56CD14). DC subsets were then identified as myeloid (HLA-DR+CD11c+) or plasmacytoid (HLA-DR+CD11c, with further confirmation as CD123+). B,C. Plots show the differential expression of TNF-α and IFN-α (x- and y-axis respectively) within the pDC (blue) and mDC (pink) subsets in unstimulated cells (left column) as well as cells stimulated with LPS, R837 and R848. Unstimulated cells expressed neither cytokine, while mDCs expressed TNF-α when stimulated with LPS or R848, and pDCs expressed TNF-α when stimulated with R837 or R848. Only pDCs stimulated with R848 expressed IFN-α.


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