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Creative Biolabs has accumulated rich experience in the field of single-cell to help you plan and optimize your single cell death analysis experiments.

Cell Death

Cell death plays an essential role in the development of organisms, the etiology of disease, and the reactions of cells to therapeutic treatments. Apoptosis is a type of programmed cell death that involves caspases, which are specialized cysteine proteases found as inactive proenzymes in animal cells. From the standpoint of basic research, apoptosis is a classic systems-level challenge in which complicated circuits comprising graded and competing chemical signals dictate binary life-death decisions at the single-cell level. From a clinical standpoint, diseases like cancer cause a break in the natural balance between cell proliferation and cell death, and anticancer medications are thought to work by inducing apoptosis in cancer cells.

Simplified schematic of receptor-mediated apoptosis signaling.Fig.1 Simplified schematic of receptor-mediated apoptosis signaling. (Spencer, 2011)

Single-Cell Analyses Using Real-Time Kinetic Labeling

Characterizing perturbagens employed as research tools and in drug discovery pipelines requires quantifying cytostatic and cytotoxic consequences. Furthermore, to properly analyze the function and impact of these perturbagens, data-rich acquisition combined with strong analysis methodologies is required. A method for single-cell and population-level analyses using real-time kinetic labeling (SPARKL) was developed. SPARKL combines high-resolution live-cell imaging with automated detection and analysis of cell death fluorescent reporters. SPARKL is more sensitive, accurate, and high throughput than traditional methods of detection and analysis, while also removing sample processing and delivering richer data.

Comparison of workflows for cell death analyses using flow cytometry or SPARKL.Fig.2. Comparison of workflows for cell death analyses using flow cytometry or SPARKL. (Gelles, 2019)

Design of SPARKL

SPARKL uses fluorescently tagged annexin V (AV) because of its stability, signal strength and duration, stoichiometric binding, and importance to cell death machinery. Scientists evaluate and verify a variety of fluorescent labeling reagents that are suitable for long-term incubation and time course research, as well as show how they can be utilized to investigate elements of cell death kinetics. They further add to the versatility by providing various examples of adaptations and mathematical analysis to dig deeper into the kinetic data.

Workflows of SPARKL.Fig.3. Workflows of SPARKL. (Gelles, 2020)

Advantages of Our Service

  • Visualize and quantify single cell death using imaging
  • Automatic analysis of the time course of cell death.
  • Single cell death analysis by flow cytometry
  • Multiplex assessments of proliferation and apoptosis

Published Data

Paper Title Time-resolved, single-cell analysis of induced and programmed cell death via non-invasive propidium iodide and counterstain perfusion
Journal Scientific reports
Published 2016
Abstract Prior to analysis, traditional propidium iodide (PI) staining necessitates the completion of many stages, which could alter assay results as well as cell viability. In this study, through live-cell imaging during perfusion with 0.1M PI inside a microfluidic culture device, this multistep analytical method was changed into a single-step, non-toxic, real-time method. The use of dynamic PI staining as a live/dead analytical method yielded consistent results for single-cell death caused by direct or indirect stimuli.
Results They demonstrated a one-step, non-invasive, dynamic PI staining approach inside a microfluidic culture device that allowed for real-time monitoring of cell death events in prokaryotic and eukaryotic cells. To simplify real-time testing for survivor cells or to get further information on cell health, non-toxic counterstains (CALv, CALg, and PO-PRO-1) were chosen. Over the course of the experiment, the continual supply of fluorochromes in media ensured excellent distribution in dense cell cultures. As a result, appropriate fluorochrome concentrations in the M or smaller range might be achieved, minimizing non-specific cell coloring caused by fluorochrome uptake at large concentrations.

Determination of optimal propidium iodide concentration.Fig.4 Determination of optimal propidium iodide concentration. (Krämer, 2016)

Creative Biolabs is a trusted partner to offer custom single cell death analysis services for our global customers. Please contact us for more information. Our experts will help design an optimal solution for your project and trouble-shoot for you throughout the whole process.

References

  1. Spencer, S.L., Sorger, P.K. Measuring and modeling apoptosis in single cells. Cell. 2011; 144(6): 926-939.
  2. Gelles, J.D., et al. Single-cell and population-level analyses using real-time kinetic labeling couples proliferation and cell death mechanisms. Developmental cell. 2019; 51(2): 277-291. e4.
  3. Gelles, J.D., Chipuk, J.E. High-throughput cell death assays with single-cell and population-level analyses using real-time kinetic labeling (SPARKL). STAR protocols. 2020; 1(1): 100034.
  4. Krämer, C.E.M, et al. Time-resolved, single-cell analysis of induced and programmed cell death via non-invasive propidium iodide and counterstain perfusion. Scientific reports. 2016; 6(1): 1-13.
! ! For Research Use Only. Not for diagnostic or therapeutic purposes.

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