BD Accuri News

 

December 2016


Spotlight

Mirko Corselli on testing the BD Accuri™ C6 Plus

Corselli Spotlight - Thumb

As a supervisor in the Application Support group at BD Biosciences, Dr. Mirko Corselli develops applications that showcase how flow cytometry can enable investigation and discovery in different research areas. Dr. Corselli spoke to us about the range of applications he’s worked on at BD, and described his experience as an alpha tester for the new BD Accuri™ C6 Plus flow cytometer.
Read the interview »

Application Highlight

Understanding and exploiting endogenous CRISPR-Cas systems

CRISPR-Cas systems have achieved such notoriety as a genome editing tool that we sometimes forget that they evolved as endogenous prokaryotic immune systems, and their potential usefulness extends in many directions. In a recent BD Biosciences webinar on molecular biology applications of personal flow cytometry, Dr. Chase Beisel of North Carolina State University described his laboratory’s research program to understand CRISPR-Cas biology and exploit CRISPR-Cas systems. He also explained why he and his colleagues are “heavy users” of their BD Accuri™ flow cytometer.

As Dr. Beisel explained in our 2015 interview, CRISPR-Cas systems are naturally adaptive immune systems found in bacteria and archaea. Their role is to fend off foreign invaders like plasmids and bacteriophages. Unlike our own adaptive immune systems, they use RNA as the recognition element, looking for complementary nucleic acids—primarily DNA. When it finds them, it instructs the system to cleave and degrade the DNA. It's a great way to get rid of these invaders.

In the webinar, Dr. Beisel explored Type I CRISPR-Cas systems, which rely on multiple proteins and have a unique ability to cleave and degrade DNA. (They are far more common in nature than the Type II CRISPR-Cas system and its Cas9 protein used in genome editing.) For example, Type I-E CRISPR-Cas systems found in E. coli use a Cas protein complex called Cascade to process the CRISPR array, use the RNA to bind to target sequences, and then recruit Cas3 to nick and degrade the displaced DNA strand. The Beisel lab showed that eliminating Cas3 still allowed Cascade to bind its DNA target, resulting in blocked transcription at that site.

Figure 1 shows the workflow for an experiment that attempted to co-opt this system as a gene silencing method. A BD Accuri flow cytometer was used to measure GFP as a reporter of gene expression.

Color Gate Tips and Tricks - Fig 3 Large
Figure 1. Workflow for transcriptional repression studies
Figure courtesy of Dr. Chase Beisel, North Carolina State University

The results are shown in Figure 2. Tn and NTn represent the different locations at which transcription was targeted by blocking RNA polymerase. Flow cytometry analysis of GFP expression showed up to 100-fold (or more) repression of gene expression compared to a non-targeting control, primarily when targeting the promoter (as opposed to coding regions). The histograms at bottom right, output from BD Accuri™ software, show differential repression for three representative targeting locations. Gene silencing was uniform (rather than bimodal) across the population—a flow cytometry finding that would not have been possible using bulk characterization techniques.

Gene Expression App Highlight - Fig 2 Thumb
Figure 2. Gene suppression using a Type I CRISPR-Cas system in E. coli
Gene Expression App Highlight - Fig 2 Large
Figure 2. Gene suppression using a Type I CRISPR-Cas system in E. coli

A modified Type I CRISPR-Cas system was used to arrest transcription at one of several gene sites shown at top. The graph at lower left shows fold repression at each site, measured by a GFP reporter. The flow cytometry histograms at lower right show representative repression at different locations. Data courtesy of Dr. Chase Beisel, North Carolina State University.

In the webinar, Dr. Beisel presented other experiments designed to explore and exploit CRISPR-Cas systems. In addition, BD scientist Dr. Mirko Corselli presented data on the use of flow cytometry to optimize transfection efficiency in mammalian cells and to detect fluorescent proteins in bacteria. From both presentations, it was clear that personal flow cytometry is an invaluable tool in molecular biology applications.

For a limited time, you can take advantage of a promotion that brings flow cytometry even more within reach. Purchase a new BD Accuri™ C6 Plus flow cytometer today and get an immediate $5,000 discount. Or, purchase the BD Accuri C6 Plus flow cytometer with the BD CSampler™ Plus automation accessory and receive a total immediate discount of $10,000. Finally, add the Selectable Laser Module, which expands the range of fluorochromes you can analyze, and get $5,000 worth of reagents free from BD.

Replay the webinar »

Download our product information sheet on molecular biology applications »

Check out the promotion »


 

Tips & Tricks

Use color gating to add visual contrast

Color Gating Tips and Tricks - Fig 3 Thumb
Figure 3. Color gating on the BD Accuri C6 Plus
Tips & Tricks - Test Compounds - Large
Figure 3. Color gating on the BD Accuri C6 Plus

A. Lymphocytes were identified and gated using light scatter. B. JNK cells were identified and gated as CD16+CD3 and colored red using BD Accuri C6 Plus software.C. NK cells are now colored red in the original light scatter plot.

The new BD Accuri C6 Plus includes software support for color gating, which allows you to designate all events within one or more regions to appear in a color you specify. The color will appear in other histograms and dot plots, and will automatically update during data acquisition.

Figure 3 shows a light scatter plot (FSC x SSC) in which lymphocytes are identified in an elliptical gate. In the middle plot, NK cells are identified by their CD16 and CD3 expression, gated, and colored red. Now, in the light scatter plot, the NK cells appear red in the light scatter plot.


 

Publication Picks

This section highlights interesting recent research articles using BD Accuri flow cytometers.

Tumor cell enrichment

Lapin M, Tjensvoll K, Oltedal S, et al. MINDEC–An enhanced negative depletion strategy for circulating tumour cell enrichment. Sci Rep. 2016;6:28929. PubMed

New sepsis therapy

Cho EJ, Doh KO, Park J, et al. Zwitterionic chitosan for the systemic treatment of sepsis. Sci Rep. 2016;6:29739. PubMed

Malaria treatment

Goh, YS, Peng K, Chia WN, et al. Neutralizing antibodies against Plasmodium falciparum associated with successful cure after drug therapy. PloS One. 2016;11:e0159347. PubMed

Scorpion venom

Rigoni, VL, Kwasniewski FH, Vieira RP, et al. Human bronchial epithelial cells injury and cytokine production induced by Tityus serrulatus scorpion venom: An in vitro study. Toxicon 2016;120:22-28. PubMed


Class 1 Laser Product.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.

1 Program currently open to scientists and research labs in the US and selected European countries only.