BD ACCURI NEWS
Water quality update
Maintaining the quality of our drinking water is critical. Yet conventional plate-based methods of water testing take too much time and miss viable but non-culturable (VBNC) microbes. Flow cytometry is increasingly being used to provide near-real-time (or even real-time) analysis of drinking water. We checked in with two sets of previous BD Accuri News interviewees whose recent publications highlight these advances.
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Pick your own favorite offer for the BD best-in-class multicolor flow cytometer
With up to three lasers, the BD FACSCelesta™ flow cytometer is the brilliant multicolor solution for dim signal and rare cell populations, setting the standard for multicolor flow cytometry. For a limited time, choose the Big, Bigger or Biggest discount on the BD FACSCelesta and save.
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An unprecedented special offer on the BD Accuri C6 Plus
The BD Accuri™ C6 Plus personal flow cytometer, together with BD reagents, delivers something that every researcher should be afforded: the power of personal flow cytometry. For a limited time, you can purchase the BD Accuri C6 Plus today with an immediate $7,000 discount. Or, purchase the BD Accuri C6 Plus with the BD CSampler™ Plus automation accessory and receive a total immediate discount of $15,000.
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Microbiology research with the BD Accuri C6 Plus
In the diverse field of microbiology research, flow cytometry is a versatile and powerful technique for analyzing microorganisms, including bacteria and yeast. Light scatter data can reveal basic information about microbes' size, shape and surface features, while fluorescent stains can assess their cell viability, metabolic activity and concentration. The BD Accuri C6 Plus can provide data for a range of microbiological applications, such as measurement of gene expression, monitoring bacterial and yeast fermentations, recombinant protein production in bacteria, environmental research, food processing and monitoring drinking water.1
Perhaps the most common task in microbial analysis is to identify and count microbes. The unique fluidics system of the BD Accuri C6 Plus, driven by peristaltic pumps, allows it to determine sample volume and count cells rapidly, directly and automatically from the software, eliminating laborious plate counts.
SYBR® Green is a cell-permeable DNA dye often used as a total cell stain to tag bacteria in environmental samples such as drinking water. This approach, called fluorescence triggering, provides increased separation of SYBR® Green+ bacteria from background, compared to the use of light scatter to identify bacteria based on size. When the cell-impermeable DNA dye propidium iodide (PI) is added to the staining protocol, it can discriminate SYBR® Green+PI– viable bacteria (with intact membranes) from SYBR® Green+PI+ damaged or dead bacteria (with disrupted membranes). This assay, which can be streamlined using a free downloadable software template, was standardized by Frederik Hammes and his colleagues at Eawag, whose work is profiled in this month’s Spotlight feature
Figure 1 shows co-staining of an E. coli culture with SYBR® Green and PI. When treated with increasing concentrations of ethanol, damaged bacterial cells shift from the Live to the Dead cell gate on the FL1 vs FL3 plot. The line graph shows nearly identical results when the cells are counted directly by volumetric counting on the BD Accuri C6 Plus, compared with the use of counting beads.
Researchers analyzing aquatic samples on the BD Accuri C6 Plus can identify the broad categories of phytoplankton using their characteristic fluorescence profiles, reflecting their relative production of chlorophyll, phycocyanin and phycoerithrin (PE), detected in FL3, FL4 and FL2 respectively (Table 1).
These naturally produced fluorophores allow researchers to use flow cytometry to directly detect, discriminate, analyze by morphology and enumerate these organisms using fluorescence-detection methods on BD Accuri C6 Plus, without the addition of extraneous dyes or probes. You can see an example in last month’s interview with BD scientist Mirko Corselli.
Compact size, rugged design and portability make the BD Accuri C6 Plus ideal for environmental microbiological research in the field. Fixed optics and capillary sheath flow fluidics enable continuous operation, even during motion and vibration. BD Accuri™ flow cytometers have traveled to field sites across the globe, from the peaks of the Himalaya to the forests of China, from the Great Lakes to the Gulf of Finland and from the Arctic to the Antarctic.
Tips & Tricks
Use BB515 to detect dim antigens
The popular fluorochrome FITC is not always bright enough to detect dim antigens. A brighter alternative is BD Horizon Brilliant™ Blue 515 (BB515). Its excitation peak exactly matches the 488-nm laser in BD Accuri flow cytometers, and it emits a narrower band of fluorescence that is detected in the FL1 channel.
Figure 2 shows the results of two different regulatory T-cell (Treg) panels in which the dim antigen CD25 was paired with either FITC (upper plots) or BB515 (lower plots). In the dot plots on the left, the mean fluorescence intensity (MFI) of the cells stained with BB515 was shifted right, indicating brighter staining. In the histograms on the right, BB515 was able to resolve a CD25mid population that FITC did not detect.
This section highlights interesting recent research articles using BD Accuri flow cytometers.
New melanoma therapy
Alderman C, Sehlaoui A, Xiao Z, Yang Y. MicroRNA-15a inhibits the growth and invasiveness of malignant melanoma and directly targets on CDCA4 gene. Tumor Biol. 2016;3713941-13950. PubMed
Liu X, Zeng B, Zhang J, et al. Role of the gut microbiome in modulating arthritis progression in mice. Sci Rep. 2016;6:30594. PubMed
Telomerase RNA analysis
Xu J, Wang Y, Yang L, Gao Y, Li B, Jin Y. A cytometric assay for ultrasensitive and robust detection of human telomerase RNA based on toehold strand displacement. Biosens Bioelectron. 2017;87:1071-1076. PubMed
CRISPR-Cas genome editing
Benakanakere M, Finoti LS, Tanaka U, Grant GR, Scarel-Caminaga RM, Kinane DF. Investigation of the functional role of human Interleukin-8 gene haplotypes by CRISPR/Cas9 mediated genome editing. Sci Rep. 2016;6:31180. PubMed
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.
1All reagents and kits are compatible with both the BD Accuri™ C6 and the new BD Accuri C6 Plus flow cytometer systems. Data was generated on the BD Accuri C6 Plus. Information about BD reagent kits, BD Accuri™ C6 and BD Accuri™ C6 Plus software templates, and BD CSampler™ and BD CSampler Plus automation options is available at www.bdbiosciences.com/accuri.
Cy™ is a trademark of GE Healthcare. Cy™ dyes are subject to proprietary rights of GE Healthcare and Carnegie Mellon University, and are made and sold under license from GE Healthcare only for research and in vitro diagnostic use. Any other use requires a commercial sublicense from GE Healthcare, 800 Centennial Avenue, Piscataway, NJ 08855-1327, USA.
SYBR® is a registered trademark of Life Technologies Corporation.