BD Accuri C6

 

Algal Biofuels

Light-scatter profiles for microalgae of varying size
Light-scatter profiles for microalgae of varying size
A. FSC-A histograms for 11 species of microalgae, ranging in size from 2 to 15 µm, are overlaid across the entire dynamic range of the BD Accuri™ C6 (left) and “zoomed” for easier comparison (right).
B. FSC-A vs SSC-A plots of the smallest, largest, and an intermediate species (A, F, and H) using logarithmic axis scaling. The maximum channel values of the axes are identical for each plot (FSC-A=1.25 x 106, SSC-A=2.0 x 106). Growth conditions for each strain were a 12-h light:12-h dark photoperiod, 100 µE/m2/s, and 20°C. Cultures were sampled and analyzed using Medium fluidics speed (core size 16 µm) at the transition from logarithmic to stationary growth phase. Ten thousand events were measured for each sample and gated to exclude debris.
Data courtesy of James Barker and Rose Ann Cattolico, University of Washington.
Neutral lipid content in Isochrysis sp., analyzed with Nile Red
Neutral lipid content in Isochrysis sp., analyzed with Nile Red
Isochrysis cells, either (A) unstained or (B) stained with 1 µg/mL of Nile Red, were analyzed on a BD Accuri C6 to detect neutral lipid content. Nile Red is intensely fluorescent in organic solvents and hydrophobic environments. Data is plotted on 2D density plots of chlorophyll fluorescence (x-axis: FL3; ex: 488 nm, em: 670 nm LP) vs Nile Red fluorescence (y-axis: FL2; ex: 488 nm, em: 585 ±20 nm). Staining resulted in a 200-fold increase in FL2 signal, while the increase in FL3 fluorescence (chlorophyll a autofluorescence) was minimal.
C. Data from unstained (black) and stained (red) cells are plotted on a single-parameter histogram (Nile Red; FL2). Debris was excluded from the analysis by gating.
Data courtesy of Gordon Wolfe, California State University (Chico).

 
 

Apoptosis, Cell Cycle, and Proliferation

Flow cytometric analysis of apoptotic and non-apoptotic populations for active Caspase-3
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.
Apoptosis analysis using the FITC Annexin V kit and software template
Apoptosis analysis using the FITC Annexin V kit and software template
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. Using the BD Pharmingen™ Annexin V FITC Apoptosis Detection Kit (Cat. No. 556570), cells were stained with Annexin V FITC and PI according to the kit staining protocol, acquired on a BD Accuri™ C6 flow cytometer using the kit template, and analyzed using BD Accuri™ C6 software. Results: Camptothecin treatment (lower plots) resulted in an increase in early apoptotic cells (PIAnnexin V+, shown in green) compared to the DMSO control (upper plots). Dead or late-stage apoptotic cells (PI+Annexin V+, red) and live cells (PIAnnexin V, black) were easily distinguished.
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. 559619 or 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.
Flow cytometric analysis of PE Annexin V staining
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.
Example: proliferation study
In vitro T-cell proliferation assay

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

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.

 
 

Bead-Based Immunoassays

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

 
 

Bioprocess Monitoring

Viability and vitality of E. coli cells following chemical stress
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 (BOXPI) 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.

 
 

Cell Counting

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).

 
 

DNA Analysis

Distinguishing ploidy in A. thaliana root tissues
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
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).

 
 

Gene Expression

Optical filter set comparison table

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 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 multiple fluorescent proteins

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

Fluorescent protein detection - Transfection screening

Fluorescent protein detection - Transfection screening


Detection of GFP expression in bacteria
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
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.

 
 

Immunology

T-cell phenotyping, 4-color analysis
T-cell phenotyping, 4-color analysis

Thawed human PBMCs were stained with directly labeled anti-CD45RA FITC, CD4 PE, CD8 PE-Cy™7, and CD3 APC in PBS + 1 mg/mL of BSA, covered, on ice, for 30 minutes. Cells were acquired and gated on lymphocytes to identify CD3+CD8+ cytotoxic (blue) and CD3+CD4+ helper (red) T-cell populations.

After FITC staining, subpopulations of CD45RA+ and CD45RA cytotoxic and helper T cells were identified.

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.

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).
BD Pharmingen™ Human Naïve/Memory T Cell Panel (Cat. No. 561438) analysis on the BD Accuri C6
BD Pharmingen™ Human Naïve/Memory T Cell Panel (Cat. No. 561438) analysis on the BD Accuri C6
Human peripheral blood was 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. (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 (CD45RACD197+, red), or effector memory cells (CD45RACD197, purple).
Analysis of T-cell cytokines using the intracellular phenotyping kit and software template
Analysis of T-cell cytokines using the intracellular phenotyping kit and software template
Using the BD Pharmingen™ Human Th1/Th2/Th17 Phenotyping Kit (Cat. No. 560751), purified human PBMCs were stimulated with PMA/Ionomycin (at 50 ng/mL and 1 μg/mL, respectively) in the presence of 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.

 
 

Intracellular Calcium

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.

 
 

Microbiology

Determination of bacterial strain by PNA FISH

Determination of bacterial strain by PNA FISH
Method: Determination of bacterial strain using S. aureus PNA FISH® reagents (AdvanDx, Inc.).
Resolution of four microbial populations using light scatter and an autofluorescence trigger
Resolution of four microbial populations using light scatter and an autofluorescence trigger
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

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
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.
Live/dead discrimination of E. coli using the BD™ Cell Viability Kit
Live/dead discrimination of E. coli using the BD™ Cell Viability Kit

E. coli cells were grown in LB broth overnight and treated with Conflikt® Detergent Disinfectant (1%) at room temperature for 5 minutes to induce cell death. The treated and untreated samples were stained with the BD Cell Viability Kit (Cat. No. 349483) and acquired on a BD Accuri™ C6 flow cytometer for 30 seconds on the Fast flow rate (66 µL/min) with SSC-H threshold = 10,000 to exclude debris. Results: A. Cells were initially gated on an FL2-A vs SSC-A plot as described in the product insert. B, C. Simultaneous staining with thiazole orange (TO) and propidium iodide (PI) allows distinction among live (TO+PI), dead (TO+PI+), and injured (TO+PIint) cell populations, revealing increased cell injury and death in the treated sample as expected. The TOPI+ population was excluded from the analysis as debris.

Conflikt is a registered trademark of Decon Labs, Inc.


 
 

Phagocytosis

Example: phagocytosis study

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

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. 560477), H9 human embryonic stem cells (hESCs) were disassociated using BD™ Accutase™ Cell Detachment Solution (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.

 
 

Miscellaneous

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



For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Class 1 Laser Product.
Alexa Fluor® is a registered trademark of Molecular Probes, Inc.
Conflikt is a registered trademark of Decon Labs, Inc.
Cy™ is a trademark of Amersham Biosciences Corp. Cy™ dyes are subject to proprietary rights of Amersham Biosciences Corp. and Carnegie Mellon University and are made and sold under license from Amersham Biosciences Corp. only for research and in vitro diagnostic use. Any other use requires a commercial sublicense from Amersham Biosciences Corp., 800 Centennial Avenue, Piscataway, NJ 08855-1327, USA.
FlowJo is a trademark of Tree Star, Inc.
PNA FISH is a registered trademark of AdvanDx, Inc.