The BD FACSCelesta flow cytometer is designed to make multicolor flow cytometry more accessible and allow researchers to benefit from new innovations in instrument and reagent technology. Parallel advances in optical and reagent technology are pairing multilaser, multidetector instruments with bright new dyes, thereby enabling increasingly deep and powerful insights in cell analysis.
The BD FACSCelesta platform offers four configurations, each of which is optimized to enable the use of traditional as well as innovative BD Horizon Brilliant™ dyes that minimize spectral overlap and simplify experimental design and analysis for new and experienced researchers. The system also allows you to gain access to the advanced reagent technology of the BD Horizon Brilliant dyes that can help you detect low-density antigens and rare populations. The BD FACSCelesta cell analyzer has been designed to fit on the benchtop and leverages proven BD FACSDiva™ software to streamline workflow from system setup to data acquisition to data analysis.
Dr. Nu Zhang on tissue-resident memory T cells
Dr. Nu Zhang is an assistant professor at the University of Texas Health Science Center in San Antonio, TX. His research focuses on tissue-resident memory T (T RM) cells, a recently identified population that plays a major role in adaptive immune surveillance. Dr. Zhang spoke to us about why T RM cells are important, what he's learned about them using a new BD FACSCelesta flow cytometer and his recent paper published in the journal Nature (see below).
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Benjamin J. Daniel discusses the popularity of the BD FACSCelesta™ flow cytometer in his core facility
Benjamin J. Daniel, PhD, is the director of the Institutional Flow Cytometry Facility and a research assistant professor at the UT Health, San Antonio, TX. He is also vice president of FlowTex, a Texas-based annual flow cytometry conference, and has served on the program committee for the past several CYTO conferences. Dr. Daniel told us why a new 3-laser BD FACSCelesta™ flow cytometer quickly became popular at his core facility, and described the various research groups that use it.
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Publications Citing the BD FACSCelesta
Ma C, Mishra S, Demel EL, Liu Y, Zhang N. TGF-β controls the formation of kidney-resident T cells via promoting effector T cell extravasation. J Immunol. 2017;198:749-756. PubMed
Hallowell RW, Collins SL, Craig JM, et al. mTORC2 signalling regulates M2 macrophage differentiation in response to helminth infection and adaptive thermogenesis. Nat Commun. 2017;8:14208. PubMed
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.
Cell cycle analysis on the BD FACSCelesta BVR configuration
Bromodeoxyuridine (BrdU) is an analog of the DNA precursor thymidine. When cells are incubated in the presence of BrdU, the molecule is incorporated into newly synthesized DNA by cells entering and progressing through the S phase of the cell cycle. It can then be detected with antibodies against BrdU. To determine the amount of total DNA, samples can also be stained with a DNA dye such as DAPI. With this combination, two-color flow cytometry can determine the cell cycle phase (G 0/G 1, S, or G 2/M) of cells that are actively synthesizing DNA. In this analysis, cells were gated based on light scatter. Cells pulsed with BrdU (lower plots) show a characteristic horseshoe pattern.
CS&T beads ensure data consistency
Analysis of BD FACSDiva CS&T research beads (gated on forward vs side scatter signals) shows the separation between dim, medium, and bright beads for each fluorescence channel on the BD FACSCelesta. After each daily setup and QC run, BD FACSDiva automatically adjusts PMT voltages to ensure consistent results from day to day and across instruments. BD FACSDiva CS&T research beads are excited by all supported lasers and emit in the range of virtually any filter combination.
Expression of fluorescent proteins in transfected human embryonic kidney cells
HEK-293 cells were transfected over 24 hours with AcGFP (left) or mCherry (right), fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655) and cryopreserved for one week. After thawing, cells were washed and then analyzed by flow cytometry. Transfected cells (green or red) were compared to wild type (black) for expression of AcGFP or mCherry, respectively.
Rainbow beads demonstrate sensitivity and resolution
SPHERO™ 8-Peak Rainbow Calibration Particles (RCP-30-5A) are calibration particles designed to measure the performance of fluorescence channels from 365 to 650 nm. The narrow bands and clear peaks demonstrate the sensitivity and resolution of the BD FACSCelesta for three compatible fluorochromes.
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 +
helper T cells and finally on CD25 +
Tregs to identify naïve (CD45RO -
) and memory (CD45RO +
) 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.