Cflow Plus Analysis Essay

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Overview

Cellular processes and events are probabilistic and take place in a context of complex varying environments, which they influence and which influence them. For this reason, flow cytometry is an ideal methodology for cell and cancer biology research since it supports the multiparametric analysis of individual cells and subpopulations in heterogeneous samples.

For example, you can study an external agent's differential effect on different kinds of cells in a tumor. You can analyze cell cycle and aneuploidy or trace signaling cascades across multiple subpopulations. Results are rapid and resolve data at the single-cell level rather than averaging across a sample.

A personal flow cytometer in the lab provides many advantages for cell and cancer biology studies. When cells are ready for analysis or rare tumor samples arrive, it's crucial to have a flow cytometer at hand, ready to go. With two lasers and four fluorescence detectors, BD Accuri personal flow cytometers bring this versatility and convenience to your benchtop. Two additional features can further simplify your research:

Absolute counts. Cell concentration can be calculated directly from BD Accuri™ software statistics tables without the addition of counting beads.

Kits and templates. BD Biosciences offers a range of cost-effective cell biology kits ideally suited for the BD Accuri that include needed reagents and buffers to assess apoptosis, cell cycle and proliferation, and DNA damage. Free BD Accuri software templates simplify data collection and analysis.

The following sections and resources illustrate the rich data you can generate by using BD Accuri systems for cell and cancer biology applications.

Resources


Apoptosis, Cell Cycle, DNA Damage, and Proliferation

Many factors, including stress, radiation, environmental exposure, and treatment with small molecules, can lead to changes in cell cycle, apoptosis, DNA damage, and cell proliferation. Detecting these events has broad application in the fields of cell biology, cancer research, and developmental biology.

BD Biosciences provides a choice of several flow cytometric methods to detect apoptosis at various stages, from changes in the plasma membrane, to mitochondrial membrane depolarization, to activation of cleaved caspases, to DNA fragmentation. Apoptotic cells can be detected alone or simultaneously along with DNA damage and cell proliferation.

Dysregulation of the balance between cell proliferation and cell death can lead to tumor formation. Therefore, understanding the molecular signals that regulate cell cycle and apoptosis is key to the advancement of cancer research. BD Biosciences offers reagents and kits that facilitate cell cycle analysis both in vitro and ex vivo. These DNA intercalating agents and nucleotide analogs allow researchers to measure cellular DNA content, separate cells into G0/G1, S, and G2/M phases, analyze DNA ploidy, and detect DNA synthesis in actively dividing cells.

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Sample Data

Cancer Research

Immunophenotyping is one of the foremost applications of flow cytometry due to its ability to recognize different cell types based on the expression of surface and intracellular proteins. In addition to apoptosis, cell cycle, and proliferation studies, immunophenotyping is essential for understanding the cellular ecology of cancer cells.

If you don’t yet know which proteins are typically expressed by a subpopulation of interest, BD Lyoplate™ cell surface marker screening panels provide a comprehensive and efficient solution for profiling cancer cells for hundreds of human or mouse cell surface markers by flow cytometry. Deciphering the cell surface proteome enables researchers to define strategies for the analysis and isolation of targeted cells from heterogeneous populations for functional studies, drug screening, and in vivo animal studies.

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Sample Data

Cell Signaling

Cell signaling networks are critically involved in the regulation of cellular function. Cell survival, growth, and differentiation are tightly regulated through expression and post-translational modification of key proteins in signaling cascades.

Many common techniques for measuring cytokine signaling and protein phosphorylation, such as Western blot and ELISA, require cell lysis, which can not only obscure the differences among individual cells in a mixed population, but can also unintentionally activate signaling pathways. Flow cytometry provides a fast, accurate, and powerful method for investigating cell signaling. Researchers can use intracellular flow cytometry to assess production of key cytokines at the single-cell level. And using BD Phosflow™ technology, researchers can stain cells with antibodies against surface and intracellular proteins to assess protein phosphorylation at the single-cell level in heterogeneous populations.


BD Phosflow standard protocol

Resources

Sample Data

Calcium Flux

Changes in intracellular calcium levels regulate many important cellular functions. Because these changes can occur rapidly—in some cases within nanoseconds of stimulation—measuring them accurately is a significant research challenge. To add test compounds to the cell suspension, a “stop-flow” method is often used in which sampling is paused, the sample tube opened, the agonist added, and the tube resealed. This technique leaves a gap or blind spot in data collection that may fail to capture essential changes in Ca2+ levels.

BD Accuri flow cytometers employ non-pressurized peristaltic pumps in an open fluidics system. Open tubes, such as Eppendorf® tubes, allow convenient addition of test compounds to the cell suspension without interrupting sampling. This “continuous-flow” method enables nonstop monitoring of thousands of cells and accurate dynamic Ca2+ measurement of the entire population.

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Sample Data

Gene Expression

Screening thousands of cells for reporter gene expression levels is fundamental to understanding how genes are regulated inside the cell. BD Accuri systems can help you quickly examine reporter gene transfection efficiency and cell population heterogeneity—right from your benchtop. You can add additional reagents to screen cell populations, monitor DNA content, identify cells at different phases of the cell cycle, and examine heterogeneity and transient effects in reporter expression.

BD Accuri systems can measure the expression of Green, Yellow, or Red Fluorescent Protein (GFP, YFP, or RFP), as well as other fluorescent proteins, in their standard configurations. Optional filters may be used to detect signals more clearly or to resolve two fluorescent proteins with overlapping emission spectra.

Resources

Sample Data

Kinetics


Continuous-flow addition of test compounds with the BD Accuri C6

Researchers are increasingly using flow cytometry to measure kinetic cellular processes that change over time, such as cellular response to an external event or stimulus. A new method, called continuous or “real-time” flow cytometry, involves adding an agent to a sample while simultaneously analyzing it.

The real-time method can measure almost any dynamic process and is broadly applicable in cell biology and many other fields of research. Applications include cell death, erythrocyte lysis, nanoparticle uptake, platelet activation, GFP expression, optimization of staining protocols, and disinfection of drinking water. The method has the potential to dramatically increase data richness and shed light on processes that would be difficult to assess by capturing and analyzing samples at discrete intervals.

An open, non-pressurized fluidics system makes BD Accuri systems ideal for kinetic studies. Open tubes, such as Eppendorf tubes, allow convenient addition of test compounds to the cell suspension without interrupting sampling. The real-time method enables nonstop monitoring of thousands of cells and accurate dynamic measurement of the entire population.

Resources

Sample Data



All reagents and kits are compatible with both the BD Accuri C6 Plus and BD Accuri C6 flow cytometer systems. Platforms referred to as "BD Accuri" represent both the BD Accuri C6 Plus and BD Accuri C6. Data was generated on either the BD Accuri C6 Plus or the BD Accuri C6 as indicated in figure legends.

Apoptosis detection on the BD Accuri C6 Plus

Apoptosis detection on the BD Accuri C6 Plus
Annexin V and propidium iodide (PI) were used to determine the percentage of Jurkat cells entering apoptosis (green) after treatment with camptothecin (right) compared to cells treated with DMSO (left, control). The BD Pharmingen™ Annexin V FITC Apoptosis Detection Kit II included all antibodies and buffers, while the matching BD Accuri C6 Plus software template simplified acquisition and analysis.

Cell cycle analysis using BrdU flow kits and software templates

Cell cycle analysis using BrdU flow kits and software templates
Using the BD Pharmingen™ FITC or APC BrdU Flow Kit (Cat. No. 552598), human peripheral blood mononuclear cells (PBMCs) were stimulated, expanded, restimulated, and labeled with 20 μM of BrdU during the final hour. After harvesting and staining the cells with 7-AAD and either FITC or APC anti-BrdU according to the kit protocol, samples were acquired on a BD Accuri C6 flow cytometer using the kit template, and analyzed using BD Accuri C6 software. Cell cycle phases are clearly distinguished in plots showing (A) 7-AAD vs BrdU FITC and (B) 7-AAD vs BrdU APC. Cells in black (to left of G0/G1 gate) contain less DNA, which may indicate cell death.

Cell cycle and ploidy analysis on the BD Accuri C6

Cell cycle and ploidy analysis on the BD Accuri C6
K562 leukemia cells (incorporating the Philadelphia translocation) were cultured, stained with the BD Cycletest™ Plus DNA Reagent Kit (Cat. No. 340242), acquired, and analyzed on the BD Accuri C6 flow cytometer. A. Cells are gated to exclude aggregates on a PI FL2-A vs PI FL2-H plot. B. A PI histogram of the gated K562 cells distinguishes cells at the G0/G1, S, and G2/M cycle phases. C. Staining and analyzing normal PBMCs along with the K562 cells can quantify their aneuploidy by comparing fluorescence intensities of their G0/G1 peaks.

Detecting mitochondrial depolarization using BD Pharmingen™ MitoStatus reagents

Detecting mitochondrial depolarization using BD Pharmingen MitoStatus reagents
Jurkat cells (human T-cell leukemia; ATCC TIB-152) were treated with 0.025% DMSO vehicle control for 5 hours (red), 5 μM camptothecin for 5 hours to induce apoptosis (blue), or 50 μM FCCP mitochondrial uncoupler for 20 minutes (green). Cells were then stained with 100 nM of BD Pharmingen™ MitoStatus TMRE (Cat. No. 564696, upper plots) or MitoStatus Red (Cat. No. 564697, lower plots) for 15 minutes at 37°C in media, washed twice with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656), and acquired and analyzed on the BD Accuri C6 using BD Accuri C6 software. Results: A, C. Compared to the vehicle-treated controls, Jurkat cells treated with FCCP mitochondrial uncoupler show a decrease in fluorescence intensity due to depolarization of mitochondria. B, D. Camptothecin-treated cells show a mix of polarized and depolarized populations as they progress through apoptosis.

Detecting apoptosis, DNA damage, and cell proliferation using the kit and template

Detecting apoptosis, DNA damage, and cell proliferation using the kit and template
Jurkat cells (human T-cell leukemia; ATCC TIB-152) were treated with compound vehicle (DMSO) or the topoisomerase I inhibitor camptothecin (6 µM) for 4 hours at 37°C to induce apoptosis. BrdU (10 µM) was added from the BD Pharmingen™ Apoptosis, DNA Damage and Cell Proliferation Kit (Cat. No. 562253) during the last hour of treatment. The cells were harvested and stained according to the kit instructions, acquired on a BD Accuri C6 using the kit template, and analyzed using BD Accuri C6 software. Results: With DMSO treatment alone (upper plots), approximately 46.7% of the cells were proliferating (BrdU+, plot B) and very few were apoptotic (cleaved PARP+, plots B and C). The DMSO control had a low signal intensity of H2AX expression with 46.1% of the non-apoptotic cells staining positive (plot C). After treatment with camptothecin (lower plots), apoptotic cells (cleaved PARP+, plots E and F) increased and proliferating cells (BrdU+, plot E) decreased, as expected. Among non-apoptotic cells, H2AX expression increased both in percentage (73.7%) and signal intensity (plot F), indicating DNA damage.

Immunophenotyping breast cancer cell lines for cancer stem cell markers

Immunophenotyping breast cancer cell lines for cancer stem cell markers
MDA-MB-231 and MDA-MB-468 cells (human epithelial breast adenocarcinoma; ATCC) were disassociated with BD™ Accutase™ Cell Detachment Solution (Cat. No. 561527) and stained with BD Pharmingen™ Mouse Anti-Human CD24 PE and BD Pharmingen™ Mouse Anti-Human CD44 APC (Cat. Nos. 555428 and 559942). Data was acquired on a BD Accuri C6 and analyzed using BD Accuri C6 software. Results: Cells were initially gated based on light scatter properties (left plots). As expected, MDA-MB-231 cells (upper plots) expressed a cancer stem cell phenotype (CD44+CD24) while MDA-MB-468 cells (lower plots) expressed both CD24 and CD44. Gates were drawn based on isotype controls (data not shown).

Cell signaling analysis using BD Phosflow reagents

Cell signaling analysis using BD Phosflow reagents
The BD Accuri C6 Plus can examine post-translational modifications of cell signaling proteins. U-937 cells treated with IFN-γ showed increased Stat1 (pY701) phosphorylation in a dose-dependent manner. The level of phosphorylation can easily be quantified as a function of median fluorescence intensity (MFI).

Continuous sampling on the BD Accuri C6 Plus

Continuous sampling on the BD Accuri C6 Plus

Easily add external agents without interrupting data acquisition. Jurkat cells were pre-loaded with the calcium indicator BD Pharmingen™ Fluo-4 AM. Calcium levels increased immediately after treatment with the calcium ionophore A23187 (left plot), but not after treatment with DMSO control (right).

Simultaneous detection of multiple fluorescent proteins

Simultaneous detection of multiple fluorescent proteins
Simultaneous detection of GFP, mCit(YFP), and mCherry(RFP) using the alternate filter configuration.

Data generated on the BD Accuri C6.

Detection of green and yellow fluorescent proteins

Detection of green and yellow fluorescent proteins
A, C. Either GFP or YFP signal can be detected in FL1 with the standard BD Accuri C6 filter configuration (530/30 in FL1, 585/40 in FL2).
B, D. To detect both GFP and YFP at once, researchers can separate the signals by using the 510/15 filter (Cat. No. 653184) in FL1 and the 540/20 filter (Cat. No. 653528) in FL2. Top and bottom graphs show uncompensated and compensated data, respectively.

Data generated on the BD Accuri C6.

Continuous, gap-free recording of intracellular calcium levels on the BD Accuri C6

Continuous, gap-free recording of intracellular calcium levels on the BD Accuri C6
Comparative cytograms of Fluo-4 fluorescence of C6 glioma cells over time, showing the effects of adding control and test compounds (ionophore A23187, ethanol, and thapsigargin). Events are gated on high Fluo-4 fluorescence to exclude fragments. A. Upper cytograms show data obtained on a Beckman Coulter CyAn ADP using the stop-flow method, showing time gaps when compounds were added. B. Lower cytograms show data obtained on a BD Accuri C6, adding compounds in open Eppendorf tubes without interrupting sample acquisition. No time gaps were observed. Except for the gaps, comparable data were obtained by both methods. Data from Vines A, McBean GJ, Blanco-Fernández A. A flow cytometric method for continuous measurement of intracellular Ca2+ concentration. Cytometry Part A. 2010;77:1091-1097; reproduced courtesy of the authors.

Interaction of nanoparticles with mammalian cells

Interaction of nanoparticles with mammalian cells
Using continuous-flow sampling on the BD Accuri C6, fluorescent nanoparticles were successively added to mammalian cells after 1 minute and 10 minutes and measured as the increase in fluorescence in FL1 (585/40). The experiment was repeated under three different temperature conditions: at room temperature, in a 37°C water bath, and on ice. Results: A. An increase in nanoparticle fluorescence was detected almost instantaneously after both additions. B. Nanoparticle fluorescence curves were quite similar under all three temperature conditions. Both the rapid increase and the lack of temperature differences suggest that the fluorescence increases were due to attachment of the nanoparticles to the cell surface and not to nanoparticle uptake by the cells.

Continuous sampling at varying temperatures

Continuous sampling at varying temperatures
A. Nanoparticles were added during continuous sampling of cells while immersed in a 37°C water bath. B. Nanoparticles were added during continuous sampling of cells while immersed in an ice bath.

Experiment run on the BD Accuri C6.

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