How Tregs resist cell death
Kelsey Voss is a graduate student in the Emerging Infectious Diseases PhD program at the Uniformed Services University in Bethesda, MD. Her co-authored paper on FoxP3 in human regulatory T cells (Tregs) was recently published in Cellular Immunology. Ms. Voss explained why her lab’s BD Accuri™ flow cytometer is indispensable for cell death assays, and how she progressed rapidly from flow cytometry novice to teacher.
Read the interview »
Join us in Prague for CYTO
Join us at CYTO 2018 (April 28–May 2, 2018 in Prague, Czech Republic) for tutorials by two top BD scientists. Robert Balderas, VP of Biological Sciences, will review seminal technological advancements in fluorescence-assisted cell sorting (FACS) from the first commercial release to the present. Christina Chang, senior scientist, will explore new advances in multi-omics immune phenotyping, including the ability to correlate gene and protein expression at the single-cell level. And come by our booth where we’ll be featuring new technologies from BD.
Join us in Austin for AAI
We’ll be featuring the BD Accuri™ C6 Plus personal flow cytometer at AAI 2018 (May 4–8, 2018 in Austin, TX). We invite you to visit our booth where you can view a demo, talk to our scientists and ask our technical support experts questions.
Using GFP tagging to improve detection of bacteria
Suppose you are culturing bacteria in a rich medium, such as LB broth. When sampled using flow cytometry, this protein-rich medium will generate plenty of background noise. Because bacteria may be as small as 0.6 µm (compared to 4 µm for a baker’s yeast cell and >30 µm for some mammalian cell lines), they may overlap with protein and other debris in the growth medium. You can see this strong background signal in the FSC vs SSC plot of LB broth alone in Figure 1A (left plot).
You can use an FSC or SSC acquisition threshold to exclude most debris particles from the analysis. However, a more definitive method is to tag the bacteria with a fluorescent protein (such as green fluorescent protein, or GFP) and use fluorescence as a threshold or gating parameter.
When GFP-expressing E. coli are introduced into the sample in Figure 1B, its FSC vs SSC signal (left plot, orange gate) cannot be distinguished from the background noise. However, GFP fluorescence, detected in the FL1 channel, confirms that E. coli cells are indeed being detected (right plot).
To maximize the acquisition, you can raise the FSC threshold to 11,000 (Figure 1C). This excludes most of the debris particles and allows the E. coli population to be visualized and gated by scatter as well as GFP fluorescence. As a side benefit, the bacteria are acquired faster and with less data storage (because most of the noise is no longer being recorded).
Figure 1. GFP tagging improves discrimination of bacteria from debris
A 5-mL starter culture of E. coli BL21 cells was grown at 37°C in LB-Amp (1.0% tryptone; 0.5% yeast extract; 1.0% sodium chloride; pH 7; 100 μg/mL ampicillin), and inoculated into 500 mL of LB-Amp at approximately 200 cells/μL in a 2-L bioreactor. The culture was grown at 37°C and stirred at 250 RPM. Samples were acquired and analyzed on the BD Accuri C6. Results show FSC vs SSC (left) and FSC vs FL1 (GFP) density plots of (A) filtered LB medium, FSC-H acquisition threshold = 10; (B) filtered LB medium inoculated with E. coli, FSC-H threshold = 10; and (C) filtered LB medium inoculated with E. coli, FSC-H threshold = 11,000. E. coli regions were drawn based on FSC vs FL1 (GFP) plots. Data courtesy PV Peña & F Srienc.
Figure 2, excerpted from our white paper on bioprocess monitoring, shows how researchers Christian Lavarreda, Pedro Peña and Friedrich Srienc exploited GFP tagging of bacteria in a bioprocessing application. They grew a starter culture of GFP-expressing E. coli cells in an LB-based medium in a 2-L bioreactor. They used an MSP M5000 FlowCytoPrep sample preparation system to sample directly from the bioreactor and deliver the samples directly to a BD Accuri™ C6 flow cytometer. 1
Figure 2. Batch growth of GFP + and GFP –E. coli over time
E. coli BL21 cells were cultured in LB-Amp. Samples were acquired on the MSP M5000 FlowCytoPrep sample preparation system and delivered to the BD Accuri C6, automatically executing acquisition and cleaning commands. Samples were analyzed for 1.5 minutes at the Medium flow rate (35 μL/min), with an acquisition threshold of FSC-H = 11,000 to exclude debris. GFP fluorescence was detected in FL1 (533/30) using the standard emission filter. Results: A. Cell concentrations (in cells/μL, △) increased rapidly toward the end of culturing, while the percentage of GFP + events ( □) decreased. B. FSC-A vs GFP (FL1-A) density plots of samples acquired during batch growth at (left to right) inoculation, 3.3 h, 5.6 h and 7.7 h. FSC vs GFP plots display 50,000 events/sample and are gated on E. coli. Data courtesy of C. Lavarreda, PV Peña & F Srienc.
Samples were analyzed for exponential growth and GFP expression every 15 minutes following inoculation. Monitoring GFP expression over time allows discrimination between producing and nonproducing cells in a fermentation. One can tell, for example, whether reduced overall production is due to reduced production in every cell of a homogeneous population, or to a subset of low- or nonproducers. As shown in Figure 2B, the concentration of cells in the bioreactor ( △) expanded from 192 cells/µL at inoculation to 18,557 cells/μL over 8 hours of monitoring. Concurrently, GFP expression ( □) decreased over time.
Further analysis revealed a relationship between GFP expression and growth phase as shown in Figure 2B. Upon inoculation from a late-exponential/stationary overnight culture (first plot), GFP expression was high in 83.6% of cells, and the cells were relatively small (median FSC channel value = 7,635). As the cells approached their exponential growth phase (second plot), however, the GFP + population waned and a GFP – population appeared with higher FSC (median FSC signal = 18,776 GFP – vs 9,781 GFP +). This trend continued throughout the exponential growth phase, resulting in a progressive decrease in GFP + events to 1.4% of gated E. coli cells over the course of the experiment (fourth plot). Thus, two populations were observed during the exponential phase, smaller cells with high GFP fluorescence and larger cells with low GFP fluorescence. This suggested that GFP expression was induced during the late-exponential phase, and that as the cells resumed normal growth, the induction decreased until GFP expression was nearly absent.
The BD Accuri C6 Plus personal flow cytometer is ideally suited for microbiology applications such as bioprocessing. It easily detects GFP in the FL1 channel without special filters, and its volumetric counting ability can calculate cell concentrations without the use of counting beads. Its open fluidics system allows it to interface with automated sample preparation devices and continuously monitor a culture in real time. Versatile, compact and affordable, the BD Accuri C6 Plus brings the power of flow cytometry within reach for individual research labs and small institutions.
Tips & Tricks
Determine cell counts and concentrations
Because the BD Accuri C6 Plus flow cytometer can count cells and measure sample volume directly, it can simplify cell analysis by automatically calculating cell concentrations per unit sample volume. Accurate cell concentrations are essential in many research and clinical 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 the BD Accuri C6 Plus correlate highly with counting beads and are more precise than hemocytometer counts.
Several BD publications address the determination of cell counts and concentrations on BD Accuri flow cytometers. The following publication contains recommendations, tips, techniques and troubleshooting suggestions to help maximize the accuracy of cell counts and concentrations:
Download the technical bulletin, A Guide to Absolute Counting »
For special considerations when counting bacteria and other small particles, see this publication:
Download the technical bulletin, Threshold and Analysis of Small Particles »
This BD 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:
Download the white paper, Determining Cell Concentration by Direct Volume »
Finally, the following publication reports on a detailed study validating human platelet concentrations measured by direct volume without using a hematology analyzer:
Download the white paper, Platelet Counting »
This section highlights interesting recent articles that describe research using BD Accuri flow cytometers.
Mother/offspring immune defense
Keller IS, Salzburger W, Roth O. Parental investment matters for material and offspring immune defense in the mouthbrooding cichlid Astatotilapia burtoni. BMC Evol Biol. 2017;17:264 PubMed
Microorganisms in probiotics
Chiron C, Tompkins TA, Burguière P. Flow cytometry: a versatile technology for specific quantification and viability assessment of micro-organisms in multistrain probiotics products. J Appl Microbiol. 2018;124:572-584 PubMed
Interferon signaling in West Nile virus
Setoh YX, Periasamy P, Peng NYG, Amarilla AA, Slonchak A, Khromykh AA. Helicase domain of West Nile virus NS3 protein plays a role in inhibition of Type I interferon signalling. Viruses. 2017;9:doi: 10.3390/v9110326. PubMed
High-throughput screening to screen compounds
Bredemeyer AL, Edwards BS, Haynes MK, et al. High-throughput screening approach for identifying compounds that inhibit nonhomologous end joining. SLAS Discov. 2017;doi: 10.1177/2472555217746324. PubMed
Meeting – April 28–May 2, 2018 – Prague, Czech Republic
CYTO 2018 (International Society for the Advancement of Cytometry) »
Meeting – May 4–8, 2018 – Austin, TX
Immunology 2018 (American Association of Immunologists) »