BD Accuri C6 flow cytometer
Applications
Immunophenotyping
Several unique features of the BD Accuri C6 flow cytometer make it an ideal platform on which to perform no-wash, differential analysis of human peripheral blood samples.
- The fixed voltage detectors of the BD Accuri C6 simplify data collection and reduce the potential for data loss due to signal over- or under-amplification.
- The broad dynamic range of the BD Accuri C6 allows peripheral blood populations as varied in size as platelets and eosinophils to be easily analyzed in the same data file.
- The easy-to-use Zoom tool feature of BD Accuri C6 software gives the user precise control when setting gates.
- The use of volume measurement provided by BD Accuri C6 software allows calculation of the cell concentration per µL of original peripheral blood for each of five populations: platelets, lymphocytes, monocytes, granulocytes, and eosinophils.
Resources
T-cell phenotyping, 4-color analysis
Human peripheral blood mononuclear cells: T-cell phenotyping, 4-color analysis
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.
Identification and gating of five peripheral blood cell populations
Identification and gating of five peripheral blood cell populations
Gates were drawn and adjusted using BD Accuri C6 software's Zoom tool for five populations: eosinophils (eos); platelets (plat); T lymphocytes (lymph); monocytes (mono); and granulocytes (gran).
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.Cell Counting
Technology giving researchers the ability to quickly and precisely determine the absolute number or concentration of cells with a given phenotype is of great interest in diverse fields including clinical research and diagnostics, drug discovery, and cell biology. The recent advent of small, fully digital flow cytometers combines the advantages of hydrodynamically focused cell sampling, high data acquisition rates, and excellent light scatter resolution with the ability to calculate absolute counts for any identified population in a sample.
Flow cytometry provides a rapid method to identify or classify cells, although most cytometers cannot directly provide the concentration or absolute count of cells in a sample. Concentration values are generally obtained from flow cytometric data either by combining hemacytometer counts with flow cytometric population data (dual-platform counting) or by adding an internal microsphere counting standard to the flow cytometric sample (single-platform counting).
The BD Accuri C6 flow cytometer can measure volume, and therefore allows calculation of cell concentrations within BD Accuri C6 software statistics tables without the addition of counting beads. This eliminates sources of variability associated with dual platform counting and the expense of counting beads. The pre-optimized detector performance of the BD Accuri C6 flow cytometer also minimizes technician-to-technician and day-to-day variability in cytometer set-up and sample analysis.
Resources
In vitro T-cell proliferation assay
Courtesy of Reddy P, Sung Y. Department of Pediatrics, University of Michigan, Ann Arbor, MI, USA.
Validation of BD Accuri C6 flow cytometer: two parameter live vs dead cells
Method: Splenocytes from 3 to 6-month old C57BL/6 mice were irradiated with UV light. Cell viability was determined by adding 1 μL of 7-AAD from a stock solution of 1 mg/mL (BD Pharmingen™ 7-AAD staining solution) per 100-μL volume of cells.
Phagocytosis by macrophages
A. Phagocytosis of apoptotic thymocytes by macrophages. B. Mouse peritoneal macrophages (CD68+) were isolated 3–4 days post-stimulation by intraperitoneal injection of 3% thioglycollate solution. Macrophages analyzed on the BD Accuri C6 appear in the upper left quadrant of a TUNEL vs CD68 plot. C. Thymocytes (TUNEL+, for TdT dUTP nick end labeling) were isolated from mouse thymus, washed, and treated with 10 mM of dexamethasone to induce apoptosis. Apoptotic thymocytes appear in the lower right quadrant of a TUNEL vs CD68 plot. D. Treated thymocytes were incubated with primary cultured macrophages for several hours, followed by a wash to remove unabsorbed thymocytes. Macrophage cells that have absorbed damaged thymocytes (CD68+TUNEL+) appear in the upper right quadrant, while macrophage cells that have not absorbed thymocytes (CD68+TUNEL–) appear in the upper left quadrant. Data courtesy of James Shayman, MD, Department of Nephrology, University of Michigan, Ann Arbor, MI, USA.
Stem Cells
The human embryonal carcinoma (EC) cell line 2102Ep is relatively nullipotent and expresses most of the same markers as undifferentiated human embryonic stem cells (hESCs). It is useful for assay development, as a positive control, and as a reference standard to correlate data between experiments or laboratories.
The BD Accuri C6 flow cytometer is ideally suited to the analysis of EC and hESC, especially when minimizing the intra-experimental variation in flow cytometric (FC) analysis is desired. The high resolution (24-bit digital signal processing) and expanded dynamic range (greater than 6 logs for fluorescence and light scatter detectors) of the BD Accuri C6 flow cytometer allow it to function with an absolute fluorescence scale, removing the variability due to detector voltage changes. This greatly simplifies instrument setup and analysis, and allows the use of analysis templates and preset fluorescence compensation values, reducing much of the subjective, operator-dependent variation in FC analysis. The completely digital signal processing of the BD Accuri C6 flow cytometer allows the data collection and analysis phases to be separated, reducing the risk of data loss or inappropriate analysis.
Resources
Pluripotent stem cell analysis on the BD Accuri C6
Frozen fixed H9 cells were stained with antibodies contained in the BD Stemflow™ Human and Mouse Pluripotent Stem Cell Analysis Kit (Cat. No. 560477) and analyzed on the BD Accuri C6. Data shows staining of SSEA-1, SSEA-4, and Oct3/4 compared to isotype control.
