BD ACCURI C6 PLUS

Overview

Among the array of applications that facilitate immunology and stem cell studies, multicolor flow cytometry is preeminent. Immunophenotyping was one of the first applications of flow cytometry, and for over 20 years, BD Biosciences has actively supported groundbreaking research in the field with flow cytometry systems and reagents.

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. BD's solutions for cell identification, transcription factor expression, and cytokine secretion and measurement reflect a commitment to high quality and consistency needed for advanced research.

BD Accuri™ personal flow cytometers bring immunology and stem cell studies to the benchtop. With two lasers, two scatter detectors, and four fluorescence detectors, they can handle most common assays with ease. Two additional features further streamline the process:

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 immunology and stem cell kits ideally suited for the BD Accuri that include needed reagents and buffers to assess multiple cell surface and intracellular phenotyping markers. 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 immunology and stem cell applications.


 

Immunophenotyping

Flow cytometry can rapidly and accurately immunophenotype many kinds of cells, including lymphoid tissue (thymus, spleen, and lymph nodes), digested solid tissues, and blood.

For example, one common task in human blood analysis is to identify and enumerate populations of platelets, lymphocytes, monocytes, granulocytes, and eosinophils. Flow cytometry is a quick and reliable methodology for identifying these populations simultaneously in a single sample, based on their immunophenotypes (see table).

Population Immunophenotype
Platelets CD41+CD45-
Lymphocytes CD3+CD45+
Monocytes CD11b+CD14+
Granulocytes CD11b+CD14-
Eosinophils FL1++SSChigh

Several features of BD Accuri flow cytometers make them ideal platforms for differential analysis of human peripheral blood samples. First, fixed-voltage detectors simplify data collection and reduce the potential for data loss due to signal over- or under-amplification. Second, the instruments' broad dynamic range makes it easy to analyze populations as varied in size as platelets and eosinophils in the same data file. Third, the easy-to-use software Zoom tool allows precise control when setting gates. Finally, direct volume measurement allows direct calculation of the cell concentration per µL for each population of interest.

Resources

Sample Data

T-cell phenotyping, 4-color analysis

T-cell phenotyping data analyzed on a BD Accuri C6

Methods: Frozen human peripheral blood mononuclear cells were thawed quickly, washed with PBS + 1 mg/mL of BSA, and stained with appropriate antibody cocktails for 30 minutes, covered on ice. Direct monoclonal antibodies used were: CD45RA-FITC, CD4-PE, CD8-PE-Cy™7, and CD3-APC (BD Biosciences). Flow cytometric analysis was performed on the BD Accuri C6.

Identification and gating of five peripheral blood cell populations

Identification and gating of five peripheral blood cell populations
Human peripheral blood was stained and samples prepared using a red cell lyse/no-wash procedure. "Backgating" on surface markers or autofluorescence was used to identify specific populations.

Data generated on the BD Accuri C6

Using the Zoom tool to adjust gates
Using the Zoom tool to adjust gates
Human blood cells were stained and analyzed on the BD Accuri C6. A. Original polygon placement. B. Zoomed plot shows overlapping polygons. C. Adjusted polygons. D. Plot zoomed back to original scale. For method details, see the BD Biosciences technical bulletin, Identification of Human Peripheral Blood Cell Populations with the BD Accuri™ C6 Flow Cytometer (November 2011), available at bdbiosciences.com.
Differentiating naïve and memory T cells using the kit and software template
Differentiating naïve and memory T cells using the kit and software template
Human peripheral blood was stained with the BD Pharmingen Human Naïve/Memory T Cell Panel (Cat. No. 561438) according to the kit procedure, acquired on a BD Accuri C6 flow cytometer using the kit template, and analyzed using BD Accuri C6 software. (The optional drop-in reagent CD3 APC-H7 was not used.) Results: A. A gate was drawn around the CD4+ T-cell population. B. The CD4+ T cells were characterized as either naïve (CD45RA+CD197+, blue), central memory (CD45RA-CD197+, red), or effector memory cells (CD45RA-CD197-, purple).
Distinguishing Tregs with CD127 using the Treg cocktail and template
Distinguishing Tregs with CD127 using the Treg cocktail and template
Human PBMCs were stained with the BD Pharmingen Human Regulatory T-Cell Cocktail (Cat. No. 560249) according to the kit procedure. The cells were then fixed, lysed and permeabilized using the BD Pharmingen™ Human Transcription Factor Buffer Set (Cat. No. 562574) and stained with PE-conjugated anti-human BD Pharmingen™ FoxP3 monoclonal antibody (Cat. No. 560082). Samples were collected on the BD Accuri C6 flow cytometer using the kit template and analyzed using BD Accuri C6 software. CD4+ lymphocytes were identified and gated by light scatter profile and fluorescence (data not shown). Results: A. A CD25 vs CD127 plot was used to identify CD25brightCD127dim Tregs and non-Treg CD4+ cells. B, C. Tregs identified using surface markers in Panel A were validated using FoxP3 staining. CD4+ cells identified as Tregs (C) expressed higher levels of FoxP3 than did non-Tregs (B).
Identifying three lymphocyte populations using two detectors
Identifying three lymphocyte populations using two detectors
Single-stained controls were used to set up gates for (A) dead cell exclusion (PI+) and (B) lymphocytes (CD45+). C. After adding antibodies to CD19, CD3, and CD16+CD56, an FL3 vs FL4 plot clearly distinguishes CD19+ B cells (purple, UL quadrant), CD56+CD16+ NK cells (magenta, LR quadrant), and CD3+ T cells (green, UR quadrant). PE-Cy5, which has high fluorescence spillover from FL3 into FL4, was used along with APC (detected primarily in FL4) and PE-Cy7 (detected primarily in FL3) to distinguish three mutually exclusive lymphocyte populations using two detectors.

Data generated on the BD Accuri C6.

 

Cell Counting

Peristaltic Pumps - Image
The BD Accuri's peristaltic pumps

The unique fluidics system of BD Accuri flow cytometers, driven by peristaltic pumps, allows them to determine sample volume, as well as count cells, directly. This feature speeds and simplifies cell analysis by calculating cell concentrations (per unit sample volume) automatically. Accurate cell concentrations are essential in many research applications, including enumerating leucocytes, B cells, T cells, and platelets in human blood, measuring microorganism concentrations in purified water, and determining the viability of cultured cell lines. Direct counts on BD Accuri systems correlate highly with counting beads, and are more precise than hemocytometer counts.

You can download two BD Biosciences documents on cell counting from the list below. The technical bulletin contains recommendations, tips, techniques, and troubleshooting suggestions to help maximize the accuracy of cell counts and concentrations on BD Accuri systems. The white paper presents sample data and guidance for viable cell concentrations in cultured cell lines, immune cell concentrations in human peripheral blood, and platelet counts in whole, unlysed human blood.

Resources

Sample Data

Absolute cell counts measured by direct volume vs counting beads
Absolute cell counts measured by direct volume vs counting beads
Serial dilutions of Jurkat, 3T3, and U937 cells, and T cells, B cells, and platelet samples from four human peripheral blood donors, were counted on the BD Accuri C6 by two methods. X-axis values represent direct-volume measurements, while y-axis values were calculated based on counting beads. The direct counts correlate highly with counting beads (r2 = 0.9989) and are more precise than hemocytometer counts (data not shown).
 

Cytokine Analysis & Bead-Based Immunoassays

Cytokines and growth factors are a primary means of communication between cells, driving cellular differentiation that develops and supports the immune system. Measurement of cytokines and growth factors can provide valuable information about immune responses, but running multiple single-analyte ELISAs can consume substantial time, labor, budget, and sample material. Moreover, ELISAs average their results across the entire sample, obscuring differential cytokine production by different subpopulations of cells.

BD T-cell cytokine kits and reagents simplify the detection of cytokines using intracellular flow cytometry on BD Accuri flow cytometers. Intracellular flow cytometry allows the rapid analysis of cytokines and other inflammatory mediators produced by multiple, phenotypically identified subpopulations within a heterogeneous sample. It can also determine whether cytokine production by an activated cell population is the result of a few cells producing large amounts of cytokine or a large cell population producing small quantities. Finally, it can measure several cytokines simultaneously for an individual cell.

The kits include or specify transport inhibitors, buffer systems, and fluorescent antibodies needed for acquisition and analysis. Free BD Accuri software templates matched to each kit include predefined workspaces, markers, regions, gates, and parameter names for quick and easy setup and analysis.

When you need to analyze a broad panel of cytokines, consider a bead-based flow cytometric immunoassay. Like ELISA assays, BD™ Cytometric Bead Array (CBA) assays measure secreted proteins produced by entire cell populations. But BD CBA can analyze 300 beads per cytokine—the equivalent of 300 ELISA wells—for up to 30 cytokines simultaneously, using very little sample. In essence, BD CBA is equivalent to running up to 30 ELISAs at once using flow cytometry.

Resources

Sample Data

Assessing Th1, Th2, and Th17 cytokines using the phenotyping kit and template
Assessing Th1, Th2, and Th17 cytokines using the phenotyping kit and template
Using the BD Pharmingen Human Th1/Th2/Th17 Phenotyping Kit (Cat. No. 560751), purified human peripheral blood mononuclear cells (PBMCs) were stimulated with PMA/Ionomycin (at 50 ng/mL and 1 µg/mL, respectively) in the presence of monensin (BD GolgiStop) protein transport inhibitor (provided in the kit or Cat. No. 554724) for 5 hours at 37°C. Cells were stained according to the kit procedure, acquired on a BD Accuri C6 flow cytometer using the kit template, and analyzed using BD Accuri C6 software. Results: Density plots (gated on CD4+ lymphocytes) show that stimulated cells (right) were more likely than unstimulated controls (left) to produce high levels of IFN-γ, IL-4, and IL-17A as they differentiate into Th1, Th2, and Th17 helper T cells, respectively.
Assessing cytotoxic T-cell cytokines using the kit and template
Assessing cytotoxic T-cell cytokines using the kit and template
Human whole blood was drawn into heparinized tubes and stimulated with PMA (10 ng/mL) and Ionomycin (1 µg/mL) for 5 hours at 37°C in the presence of 10 ng/mL of Brefeldin A (BD GolgiPlug, Cat. No. 555029) protein transport inhibitor. Using the BD FastImmune IFN-γ/CD69/CD8/CD3 Kit (Cat. No. 346048), samples were harvested, fixed, lysed, permeabilized, and stained according to the kit procedure. They were acquired on a BD Accuri C6 flow cytometer using the kit template and analyzed using BD Accuri C6 software, gating on lymphocytes using light scatter and then on CD3+CD8+ cytotoxic T cells (A). Results: Compared to unstimulated controls (B), stimulated cells (C) were more likely to produce high levels of the activation marker CD69. Most of these CD69+ cells expressed IFN-γ as well. Data is representative of five donors.
Cytokine analysis by analyte or sample using BD CBA kits
Cytokine analysis by analyte or sample using BD CBA kits
Human peripheral blood mononuclear cells (PBMCs) were cultured for several days with plate-bound anti-CD3, soluble anti-CD27, IL-2, and IL-4. Cells were stimulated with PMA+Ionomycin for several hours before collecting culture supernatants. Samples were prepared and stained with the BD CBA Human Th1/Th2/Th17 Cytokine Kit (Cat. No. 560484), acquired on a BD Accuri C6 using the BD™ CBA Kit Accuri template, and analyzed using FCAP Array software v3.0.1. A. Bar chart of human IFN-γ levels for all standards and test samples. B. Bar chart of all seven cytokine levels for one sample.
Identification and gating of five peripheral blood cell populations
Identification and gating of five peripheral blood cell populations
Human PBMCs were cultured for several days with plate-bound anti-CD3, soluble anti-CD27, IL-2, and IL-4. In one experimental condition, cells were stimulated with PMA+Ionomycin for several hours before harvesting. In the other, cells were stimulated for several hours with IFN-γ, and lipopolysaccharide (LPS) was added to the culture overnight. All samples were prepared using the BD™ CBA Human Soluble Protein Master Buffer Kit (Cat. No. 558264) and stained with flex set reagents for 10 different cytokines. Samples were acquired on a BD Accuri C6 in the 2-blue, 2-red configuration using the BD CBA Flex Set Accuri template and analyzed using FCAP Array software v3.0.1. A. Bar chart of human IL-2 levels for all standards and test samples. B. Comparison of all 10 cytokine levels for one sample under both experimental conditions.
 

Platelets

With BD Accuri flow cytometers, research laboratories no longer need a hematology analyzer to count platelets quickly and accurately.

Until 2001, platelets were commonly counted manually using a hemocytometer or automated impedance counter, despite the serious limitations of these methods. In 2001, two professional hematology organizations introduced an International Reference Method (IRM) for platelet counting based on flow cytometry. Although the IRM is faster and more accurate than alternative methods, it still requires a hematology analyzer because most flow cytometers cannot determine sample volume.

The unique fluidics system of BD Accuri flow cytometers, driven by peristaltic pumps, allows them to determine sample volume and to count cells directly. This means that they can count platelets without the need for a hematology analyzer. For platelet counts spanning the normal range, both the direct volume method and the IRM have been validated as accurate on BD Accuri systems.

Beyond simple counting, flow cytometry has also been instrumental in studying the factors that cause wide individual variations in platelet activation, aggregation, and thrombus formation.

Resources

Sample Data

Validation of platelet counting methods on the BD Accuri C6
Validation of platelet counting methods on the BD Accuri C6

A. Correlation of platelet counts (x 109/L) obtained using the RBC/platelet ratio IRM and the direct volume method on the BD Accuri C6, and the IRM on the BD FACSCalibur™ system, for all platelet samples tested. B. Bland-Altman mean-difference analysis shows minimal bias between each pair of methods (values near zero), demonstrating that the methods are functionally interchangeable for this range of platelet samples.

In Bland-Altman analysis, the mean of the two counts for each sample is plotted on the x-axis, while their difference is plotted on the y-axis. The average difference between the two measurements is reported as the bias, with the 95% confidence interval shown in parentheses.

 

Stem Cells

BD Biosciences offers an extensive selection of hundreds of antibodies to stem cell pluripotency and differentiation markers that can be combined in many ways to monitor the cells' changing expression patterns. BD Lyoplate™ screening panels can help you discover additional surface markers that characterize cells of interest.

A primary challenge of stem cell research is the inherent heterogeneity of the cell cultures. Researchers studying embryonic stem cells (ESCs) must constantly monitor the pluripotency of their cultures, while those studying human induced pluripotent stem cells (hiPSCs) must assess reprogramming success as well as pluripotency. Researchers who study, isolate, and culture mesenchymal stromal cells (MSCs) must continually monitor and verify their purity and multipotency.

Multicolor flow cytometry is ideal for this purpose because of its ability to interrogate heterogeneous cell populations, analyze their subpopulations, and characterize multiple antigens simultaneously. Each type of stem cell or derivative expresses characteristic intracellular and surface proteins that can be used for identification. Because intracellular analysis requires permeabilization, surface marker analysis is essential when researchers want to isolate live cell populations for further analysis.

Resources

Sample Data

Stem cell analysis using the pluripotent stem cell kit and software template
Stem cell analysis using the pluripotent stem cell kit and software template
Using the BD Stemflow(tm) Human and Mouse Pluripotent Stem Cell Analysis Kit (Cat. No. 561527), stained according to kit instructions, and acquired on a BD Accuri C6 flow cytometer using the kit template. Cells were gated on light scatter properties of H9 hESCs and analyzed for expression of key pluripotency surface markers and transcription factors using BD Accuri C6 software. Results: Most analyzed cells expressed the positive pluripotency surface marker SSEA-4 and the pluripotency transcription factor Oct3/4, while few expressed the negative pluripotency marker (positive differentiation marker) SSEA-1.
Assessing iPSC pluripotency and reprogramming using the iPSC kit and template
Assessing iPSC pluripotency and reprogramming using the iPSC kit and template
Human foreskin fibroblasts (Fate Therapeutics, San Diego, CA) were reprogrammed in feeder-free SMC4 small molecule conditions.1 Specifically, CD13–SSEA-4+TRA-1-60+ reprogrammed cells were bulk sorted at Day 21 post-transduction, further expanded, and analyzed at Day 35 post-transduction. For the analysis, cells were disassociated using BD Accutase cell detachment solution (Cat. No. 561527) and fixed in 0.5X BD Cytofix™ fixation buffer (Cat. No. 554655). Then, using the BD Stemflow Human iPSC Analysis and Sorting Kit (Cat. No. 562626), they were stained according to kit instructions, acquired on a BD Accuri C6 flow cytometer using the kit template, and analyzed using BD Accuri C6 software. Results: A. Cells were distinguished from debris and gated using light scatter. B. The CD13– gate excludes the 5.0% of cells that still express markers of the starting fibroblast cell population. C. Of the gated CD13– cells, 32.9% expressed the pluripotency markers TRA-1-60 and SSEA-4. Note that cells can also be analyzed live.
1. Valamehr B, Abujarour R, Robinson M, et al. A novel platform to enable the high-throughput derivation and characterization of feeder-free human iPSCs. Sci Rep. 2012;2:213.
Verifying the MSC phenotype using the hMSC kit and template
Verifying the MSC phenotype using the hMSC kit and template
hBM-MSCs (Lonza) were disassociated using BD™ Accutase™ Cell Detachment Solution (Cat. No. 561527). Using the BD Stemflow™ Human MSC Analysis Kit (Cat. No. 562245), the cells were stained according to the kit instructions and acquired on a BD Accuri™ C6 flow cytometer using the kit template. Cells were gated on light scatter properties of hBM-MSCs and analyzed for expression of key hBM-MSC surface markers using BD Accuri™ C6 software. Results: A. Gates were drawn based on matched isotype control cocktails. B. The vast majority of analyzed cells expressed the positive hBM-MSC surface markers in the MSC positive marker cocktail (CD90, CD105, and CD73), while very few expressed the negative surface markers in the MSC negative marker cocktail (CD34, CD11b, CD19, CD45, and HLA-DR).