May 1, 2018
Nanoengineering a new injectable hemostat
Giriraj Lokhande and James Carrow are graduate students in the Department of Biomedical Engineering at Texas A&M University in College Station, TX. Their co-authored paper on nanoengineered injectable hydrogels was recently published in Acta Biomaterialia. Lokhande and Carrow explained how their new injectable hemostat can not only stop blood flow but also promote wound healing, and how their BD Accuri™ flow cytometer helps them track its performance.
Read the interview »
New poster: The dual role of immune cells in the tumor microenvironment
In the early stage of tumor growth, the innate and adaptive immune responses cooperate to identify and eliminate transformed cells (elimination phase). But if the tumor is not completely eradicated, the remaining tumor and immune cells enter a dynamic equilibrium phase in which new tumor variants continue to arise and be eliminated. This phase lasts until the tumor develops mutations that allow it to escape immune detection and elimination, eventually resulting in tumor progression (escape phase).
This complementary push-and-pull signaling of activation and inhibition has become the subject of intense scientific and therapeutic interest in immuno-oncology. Its complex mediation—by a broad array of cell types, receptors and molecules—is now illustrated visually in a new BD poster that many cancer and cell biology researchers and labs will want to post on their walls and computer screens.
On the elimination side, the poster summarizes how the immune system is alerted to the presence of tumor cells and how inflammatory signals recruit innate immune cells such as NK, NKT and γδ T cells. Dendritic cells loaded with tumor debris migrate to lymph nodes, where they activate tumor-specific CD4 + Th1 and cytotoxic CD8 + T cells. These activated T cells then migrate to the tumor site, where they join tumor-infiltrating B cells in tumor elimination. All of these cells—and more—are profiled in the poster, along with the signaling molecules they produce and respond to.
On the escape side, the poster summarizes the changes in tumor cells that result in immune escape, including loss of tumor-antigen expression, expression of ligands for co-inhibitory receptors such as PD-L1/PD1 and inhibition of phagocytosis through expression of the do not eat me signaling molecule CD47. Tumor cells can also escape immune responses by remodeling the tumor microenvironment through expression of VEGF-A and VEGF-C/D as well as other mechanisms. And tumor cells can further inhibit cytotoxic T lymphocytes through metabolic exhaustion, outcompeting them for oxygen, glucose and amino acids.
Understanding the different mechanisms by which tumor cells escape immune surveillance has driven the development of immunotherapy strategies. Such strategies are aimed at manipulating T-cell and tumor metabolism and blocking inhibitory checkpoints, immunosuppressive modulators, CD47 signaling and recruitment of immunosuppressive cells.
Tips & Tricks
Minimize spillover by design
It's well known that fluorescence spillover from one channel into another contributes to background. That's why we generally account for it mathematically in the process called compensation.
But spillover—even when compensated—also directly and irreversibly reduces the resolution sensitivity of that channel. You can see this in Figure 1. Figure 1A shows a CD4 APC vs CD8 APC-Cy™7 plot with a population of CD4 +CD8 – cells (red), a population of CD4 –CD8 + cells (blue) and a population of unstained cells (gray). Although the CD8 + cells, stained with APC-Cy7, are negative for CD4, they are spread out on the x (CD4)-axis due to spillover from APC-Cy7 into the APC channel. The spread is especially visible on an APC histogram (Figure 1B).
Now suppose there is another population of cells that express CD4, but dimly (gold oval, CD4 dim). If these cells do not also express CD8 (CD4 dimCD8 –, Figure 1C), they can readily be resolved from the unstained CD4 –CD8 – cells (gray).
But if these cells do express CD8 (CD4 dimCD8 +, Figure 1D), they cannot be resolved from the broadly distributed CD4 –CD8 + cells, even with compensation. To improve the resolution (sensitivity) of this dim subpopulation, spillover from other fluorochromes must be minimized.
Figure 1. Spillover irreversibly reduces resolution sensitivity
A. CD4 staining with APC and CD8 staining with APC-Cy7 show three distinct populations on a biparametric contour plot: CD4 +CD8 – (red), CD4 –CD8 + (blue) and CD4 -CD8 – (gray). B. The blue CD4 –CD8 + population is spread out on an APC histogram due to spillover from APC-Cy7 into the APC channel. C. A hypothetical CD4 dimCD8 – population (gold) can readily be resolved from the CD4 –CD8 – cells. D. However, a hypothetical CD4 dimCD8 + population (gold) cannot be resolved from the CD4 –CD8 + cells. Note: Data not collected on a BD Accuri™ flow cytometer.
One common strategy for minimizing spillover is to choose fluorochromes with minimal spectral overlap. For example, BD™ APC-H7 has less spillover into the APC channel than APC-Cy7—and, as an added bonus, is more stable after exposure to light and paraformaldehyde fixatives.
Four-color experiments on the BD Accuri™ C6 often use FITC, PE, PE-Cy7 and APC (detected in FL1, FL2, FL3 and FL4 respectively). This combination of fluorochromes results in modest, predictable spillover that can usually be managed with careful panel design. Of course, the requirements of your particular experiment may vary.
You can graphically view spillover for any combination of common fluorochromes in the online BD Fluorescence Spectrum Viewer. Select BD Accuri™ C6 in the Cytometer field to fill in the correct instrument specifications automatically. 1
This section highlights interesting recent articles that describe research using BD Accuri flow cytometers.
Recabarren-Leiva D, Alarcón M. Standardization of a fast and effective method for the generation and detection of platelet-derived microparticles by a flow cytometer. Immunol Lett. 2018;194:79-74. PubMed
Garlic and inflammation
Xu C, Mathews AE, Rodrigues C, et al. Aged garlic extract supplementation modifies inflammation and immunity of adults with obesity: A randomized, double-blind, placebo-controlled clinical trial. Clin Nutr ESPEN. 2018;24:148-155. PubMed
Measuring sulfur bacteria
Danza F, Storelli N, Roman S, Lüdin S, Tonolla M. Dynamic cellular complexity of anoxygenic phototrophic sulfur bacteria in the chemocline of meromictic Lake Cadagno. PLoS One. 2017;12:e0189510. PubMed
CRISPR-Cas9 mediated recombination
Antonova E, Glazova O, Gaponova A, et al. Successful CRISPR-Cas9 mediated homologous recombination in a chicken cell line. F1000Research. 2017;7:238;doi: 10.12688. Full Text
1 All reagents and kits are compatible with both the BD Accuri™ C6 Plus and the BD Accuri C6 flow cytometer systems. Data was generated on the BD Accuri C6. 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.
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