BD Accuri C6 flow cytometer

Flow cytometric analysis of apoptotic and non-apoptotic populations for active Caspase-3

Jurkat cells (human T-cell leukemia; ATCC TIB-152) were treated with 6 µM of camptothecin or 0.1% DMSO (negative control) for 4 hours to induce apoptosis. Cells were permeabilized, fixed, and stained according to the Caspase-3 kit staining protocol (Cat. No. 550914). Camptothecin treatment resulted in an increase in active Caspase-3 expression (red) compared to the DMSO control, which was primarily negative (green). Data shown was gathered on a BD Accuri™ C6 flow cytometer using BD Accuri™ C6 software.

Flow cytometric analysis of FITC Annexin V staining

Jurkat cells (human T-cell leukemia; ATCC TIB-152) were treated with 6 µM of camptothecin or 0.1% DMSO (negative control) for 4 hours to induce apoptosis. Cells were stained with FITC Annexin V and propidium iodide (PI) according to the Annexin V FITC Apoptosis Detection Kit (Cat. No. 556570) staining protocol. Camptothecin treatment resulted in an increase in early apoptotic cells (PI negative, Annexin V positive), as shown in green, compared to the DMSO control. Dead cell (PI positive, red) and live cell (Annexin V and PI negative, black) populations were easily distinguished. Data shown was gathered on a BD Accuri C6 flow cytometer using BD Accuri™ C6 software.

Flow cytometric analysis of PE Annexin V staining

Jurkat cells (human T-cell leukemia; ATCC TIB-152) were treated with 6 µM of camptothecin or 0.1% DMSO (negative control) for 4 hours to induce apoptosis. Cells were stained with PE Annexin V and 7-AAD according to the PE Annexin V Apoptosis Detection Kit (Cat. No. 559763) staining protocol. Camptothecin treatment resulted in an increase in early apoptotic cells (7-AAD negative, Annexin V positive), as shown in orange, compared to the DMSO control. Dead cell (7-AAD positive, red) and live cell (Annexin V and 7-AAD negative, black) populations were easily distinguished. Data shown was gathered on a BD Accuri C6 flow cytometer using BD™ Accuri C6 software.

In vitro T-cell proliferation assay

Courtesy of Reddy P, Sung Y. Department of Pediatrics, University of Michigan, Ann Arbor, MI, USA.

Quantitation of Mouse IL-17A on the BD Accuri C6 - Figure A

A. BD™ Cytometric Bead Array (CBA) Mouse Enhanced Sensitivity (ES) 11-plex resolved on the BD Accuri™ C6. Of the 11 cytokines analyzed, the C5 bead (labeled) corresponds to IL-17A.

Quantitation of Mouse IL-17A on the BD Accuri C6 - Figure B

B. Representative standard curve (concentration vs MFI) of the BD CBA Mouse ES IL-17A Flex Set (Cat. No. 562261) on the BD Accuri C6. After generating their own curves, researchers can calculate sample concentrations by interpolation using FCAP Array™ software.

Quantitation of Mouse IL-17A on the BD Accuri C6 - Figure C

C. Analysis of two types of mouse intracellular cytokine (MiCK-2 and MiCK-3) positive control cells using the BD CBA Mouse ES IL-17A Flex Set on the BD Accuri C6. MiCK cells express easily detectable levels of intracellular cytokines on stimulation. Different types of cells require different dilutions to obtain results within range.

MiCK-2 samples (bars on left) were prepared by stimulating purified CD4+ splenocytes with plate-bound anti-CD3, soluble anti-CD28, and recombinant IL-2 and IL-4 for two days. Cells were restimulated with IL-2 and IL-4 for three days and subsequently stimulated with PMA and ionomycin for six hours. MiCK-3 cells (bars on right) are 3-day thioglycolate-elicited peritoneal cells that have been harvested and stimulated in vitro with LPS for six hours. Samples were incubated following the instructions in the Mouse Enhanced Sensitivity Master Buffer Kit (Cat. No. 562246 or 562248), acquired on the BD Accuri C6 flow cytometer equipped with the BD Accuri Selectable Laser Module (Cat. No 653126), and analyzed using FCAP Array software v3.0.1 (Cat. No. 652099).

Selectable Laser Module increases the resolution of BD CBA Flex Set beads

A. The default configuration of the BD Accuri C6 (FL3: 670 LP; FL4: 675/25) does not adequately discriminate BD CBA Flex Set beads.
B. The BD Accuri Selectable Laser Module (Cat. No. 653126) allows reconfiguration of the BD Accuri C6 lasers and detectors, significantly expanding the fluorochrome combinations that can be analyzed. By using the module in the 2-blue/2-red setting, along with a 780/60 BP filter (included) in FL3, researchers can obtain excellent resolution for BD CBA Flex Sets.

Viability and vitality of E. coli cells following chemical stress

Biomass health includes not only viability, measured by propidium iodide (PI) exclusion, indicating an intact cell membrane, but also vitality, measured by Bis-(1,3-dibutylbarbituric acid) trimethine oxonol (BOX) exclusion, indicating a healthy polarized plasma membrane. E. coli MG1655 cells were co-stained with BOX and PI to simultaneously assess cell viability and vitality. A. A control sample was prepared by mixing live and ethanol-killed E. coli cells. Populations of live (BOX^–PI^–) and dead (BOX⁺PI⁺) cells are visible on a PI (FL3-A) vs BOX (FL1-A) plot. B. A sample harvested after the culture was subjected to chemical stress shows a third population of injured (BOX⁺PI^–) cells in the upper left quadrant. Because their cellular membranes are intact but their plasma membranes are depolarized, these injured cells stain with BOX but not PI. Data courtesy of Tim Overton, School of Chemical Engineering, University of Birmingham, UK.

Distinguishing ploidy in A. thaliana root tissues

Arabidopsis thaliana plants were germinated from seeds and grown under sterile conditions. Root tissues were chopped with a fresh razor blade, stained with propidium iodide (PI), and acquired on the BD Accuri C6. A. Nuclear contents form a linear cluster on the biparametric contour plot of FL2-A vs FL3-A fluorescence emission, corresponding to the endoreduplicative series of 2C (diploid), 4C, 8C, 16C and 32C cells. The cluster outside the rectangular region of interest (R1) contains cellular debris. B. Enlargement of the R1 region shows nucleic clusters in polygonal region P1, comprising only 1.8% of the detected events. C. Uniparametric histogram of FL2-A fluorescence, gating on region P1 of panel B, showing a near-perfect linear correlation with clear peaks. Abbreviations 2C, 4C, etc designate the appropriate C-values for the individual peaks. Data courtesy of David W. Galbraith, School of Plant Sciences and Bio5 Institute for Collaborative Bioresearch, University of Arizona, Tucson, AZ, USA. For more information, see the white paper by Galbraith and Lambert, Using the BD Accuri C6 flow cytometer for rapid and accurate analysis of the nuclear DNA contents of flowering plants (BD Biosciences, 2011).

Checking resolution with BD™ DNA QC Particles

BD DNA QC Particles (Cat No. 349523) use the DNA stain propidium iodide (PI) to validate BD Accuri C6 performance before analyzing DNA test samples. A. Chicken erythrocyte nuclei (CEN) confirm instrument linearity and resolution of singlets, doublets, triplets, and larger aggregates. B. Gating for singlets with calf thymocyte nuclei (CTN). C. Staining of CTN differentiates cells in G₀/G₁ and G₂+M phases. D. The same histogram with smaller y-axis scale not only shows the shape of the G₂+M distribution, but also reveals cells in the S phase (between G₀/G₁ and G₂+M).

Optical filter set comparison table

Listed are two filter configurations for the BD Accuri™ C6 Flow Cytometer, the recommended detector positions in which to place each filter and the fluorochromes best detected with each configuration. A variety of fluorescent proteins can be detected using the BD Accuri™ C6 Flow Cytometer, even with its standard filter configuration. However, the Fluorescence Protein Filter Set is required if simultaneous detection of GFP and YFP (or mCitrine) is needed. In addition, users detecting dim GFP signals may get increased signal resolution by using the 510/15 BP filter in place of the standard 530/30 BP. The 510/15 BP (CP170) and the 540/20 BP (CP178) are ordered as individual items, and simply swapped with the standard 530/30 BP and 585/40 BP, respectively.

Simultaneous detection of multiple fluorescent proteins

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

Simultaneous detection of multiple fluorescent proteins

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

Fluorescent protein detection

Detection of GFP expression in bacteria

Samples containing wild-type (left column) or GFP-transfected (middle column) Escherichia coli B, or a mixture of the two (right column), were acquired on a BD Accuri C6. In the mixture, transfected and non-transfected populations were resolved clearly.
Data courtesy of Tim F. Cooper, Dept. of Biology and Biological Chemistry, University of Houston, Houston, TX, USA.

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.

T-cell phenotyping data analyzed on a BD Accuri C6

Flow cytometric analysis of human peripheral blood mononuclear cells (PBMCs) using the Human Th1/Th2/Th17 Phenotyping Kit (Cat. No. 560751). Purified human PBMCs were stimulated with PMA/ionomycin in the presence of BD GolgiStop™ protein transport inhibitor for five hours. Cells were stained according to the kit procedure, acquired on a BD Accuri C6 flow cytometer, and analyzed by FlowJo™ software. Dot plots shown are gated on a CD4⁺ lymphocyte gate.

T-cell phenotyping, 4-color analysis

Thawed human PMBCs were stained with directly labeled anti-CD45RA FITC, CD4 PE, CD8 PE-Cy7, and CD3 APC in PBS + 1 mg/mL of BSA, covered, on ice, for 30 minutes. Analysis was performed on the BD Accuri C6.

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-Cy7, and CD3-APC (BD Biosciences). Flow cytometric analysis was performed on the BD Accuri C6.

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

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.

Determination of bacterial strain by PNA FISH

Method: Determination of bacterial strain using S. aureus PNA FISH® reagents (AdvanDx, Inc.).

Lake Erie water samples

Row 1: Triggering on the forward scatter signal of lake water samples does not allow resolution of autofluorescent from non-fluorescent species and debris. Row 2: Triggering on the FL3 (670-nm LP) signal of autofluorescent species enables clear resolution of at least 4 populations based on light scatter alone. Table: Accurate event counts per mL of water sample can then be determined using BD Accuri™ C6 Software.

Cyanobacterial profiling: Lake Michigan

Data prepared in collaboration with Juli Dyble and Gary Fahnensteil, NOAA Great Lakes Environmental Research Laboratory, Ann Arbor, MI, USA.

Detection of GFP expression in bacteria

Samples containing wild-type (left column) or GFP-transfected (middle column) Escherichia coli B, or a mixture of the two (right column), were acquired on a BD Accuri C6. In the mixture, transfected and non-transfected populations were resolved clearly.
Data courtesy of Tim F. Cooper, Dept. of Biology and Biological Chemistry, University of Houston, Houston, TX, USA.

Phagocytosis by macrophages

Courtesy of James Shayman, MD, Department of Nephrology, University of Michigan, Ann Arbor, MI, USA.

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.

Attenuation filters are used to bring a bright signal on scale.

Top Row: Original data showing some brightly fluorescent cells off-scale positive, using the standard FL1-A filter (530/30 BP).
Bottom Row: An attenuated (OD1) 533BP filter was placed in front of FL1 and the data re-collected.