Advanced Phenotypic Analysis



  1. PMM assays (PM-M1 through PM-M14 and PMMTox-1) are cell-based assays provided in five categories and used to determine up to 1,400 metabolic and chemical sensitivity phenotypes of mammalian cells. They use 96 well plates pre-loaded with carbon-energy and nitrogen substrates, ions, hormones/cytokines and anti-cancer agents. Cells of interest are scanned for phenotypes by adding 50 ul of a cell suspension (in IF-M1 or IF-M2 media) to each well followed by the addition of 10 ul of Biolog's proprietary redox (MA or MB) dye. In some wells, the cells are stimulated and in other wells inhibited. The generation of energy-rich NADH by the cells reduces the redox dye and brings about a color change which is then read with either a standard microplate reader or Biolog's OmniLog automated incubator-reader.
  2. Biolog's redox dyes use a new tetrazolium redox chemistry. The Biolog dyes have greatly improved sensitivity and specificity compared to previous tetrazolium dye chemistries such as MTT, XTT, and MTS.

    Biolog's dyes measure the flux of NADH production from the various catabolic pathways present in the cells being tested. The dye chemistry can transfer reducing power from the NADH inside the cell to the redox dye chemistry outside the cell.

    In many ways it is an alternative to other approaches, measuring NADH produced instead of acid produced, or oxygen consumed. The big advantage over these other approaches is that the PMM assay format is much cheaper and much more flexible.

  3. The superior redox dye chemistry enables more accurate cell growth/death studies. Substrates or metabolic effectors that enhance growth and viability, or reduce cell doubling time, or optimize bio-production yields can be readily determined.

    Model cell lines, clones, stem cells, immune cells among other cell types can be characterized and monitored based on their metabolic and chemical sensitivity phenotypes.

    In nutritional studies, PMM assays can assess the metabolic impact of gene alterations involving membrane transport systems, metabolic pathways, or cellular regulatory systems.

    In cancer research, PMM assays can be used to study the Warburg effect and the relationship between changes in oncogenes and changes in metabolism as well as shifts in sensitivity to anti-cancer drugs, hormones/cytokines, and ions.

    In mitochondrial function or toxicity assays such as chemical-agent screening programs, Biolog's PMM assays provide an extremely sensitive tool to assess mitochondrial activities.

    PMM assays are also useful for conducting QA/QC analyses of cells being developed for or used in cell-based assays and cell-based bioprocesses. They provide a simple means to identify any metabolic instability or drift, a characteristic that can significantly alter cell performance.

  4. PMM assays are applicable to all cell types, including primary cells, established cell lines, and stem cells. The only requirement is that the cell preparation allows the same number of cells to be dispensed into each well.
  5. In growing stem cells stably, it is essential to characterize the nutritional/metabolic/hormonal properties of the cells so that the key variables can be identified and controlled. PMM assays can help compare a stem cell with a cell that has lost "stemness" to see how the metabolism has changed. Unique to Biolog's redox dyes are their sensitivity at detecting cell metabolism. Depending on the cell types tested, as few as 250 cells per well can be evaluated. This is especially important when few stem cells are available for testing.
  6. The large 50 plate capacity of the OmniLog enables multiple experiments (incorporating different media formulations, cell types, or chemical agents) to be run simultaneously.

    The kinetic read capability enables the relative rates of energy flux generation (or alterations) to be assigned to each substrate or metabolic effector.

  7. The software allows differences in substrate metabolism and rates of metabolism to be quickly assessed and displayed. Each pre-loaded substrate has a KEGG database identifier number. The software allows substrates of interest to be quickly matched to their respective pathway in KEGG. Data collected over many months of experiments are stored in standard formats and can be readily retrieved and compared.
  8. Biolog's PMM assay plates can be read using a standard microplate reader. However, some of the benefits provided by the OmniLog reader will be lost.
  9. Biolog is currently developing dedicated fatty acid plates. Please contact Biolog for further information.
  10. Biolog can provide custom plates. Please contact Biolog for details.
  11. For each PMM plate production run, Biolog uses cell lines with known metabolic phenotypes to ensure the reproducibility and quality of its PMM plates.
  12. Reseachers at the Broad Institute have used PMM assays to compare metabolism in white and brown adipocytes and their precursor preadipocytes. Significant substrate utilization differences were detected. These differences could be exploited to selectively culture precursor cells or their differentiated forms.

    Researchers at Cold Spring Harbor Laboratory used the 92 anti-cancer drug PMM panel to examine drug sensitivity of cell lines with mutations commonly found in aggressive prostate cancers. The screen identified two chemicals from the PMM panel that show increased killing of the model cell lines with these mutations, suggesting new approaches to chemotherapy.

    Researchers at the Cancer Research Institute in London used the 92 anti-cancer drug PMM panel to examine colorectal cancer cells and study the anti-cancer drug sensitivity of cells with chromosomal instability. The results indicated that the cells with chromosomal instability, when compared with chromosomally stable cells, exhibited a phenotype of multi-drug resistance.

    Researchers at the Medical College of Georgia's Cancer Center studying metabolite fluxes with mass spectrometers used PMM assays to independently confirm the elevation of amino acid metabolism in prostate cancer cells treated with androgen.

  13. Uses are extremely diverse and include (1) comparison of metabolic phenotypes of activated versus un-activated T cells, (2) phenotyping cells of breast and liver cancer metastases, (3) comparison of cells from normal and gene knock-out mice to study cell-membrane nutrient transporter behavior/mechanisms, (4) assessment of the redox capacity of cancer cells (particularly related to selenoprotein function) and alterations of their sensitivities to potential or current therapeutics, (5) regulation of mitochondrial metabolism in diseases associated with mitochondrial dysfunction, (6) investigations of whether in vitro cell culture models will recapitulate well known changes in metabolic profiling activated by caloric restriction, (7) studies on p53 control of cell metabolism, (8) assessment of regulatory mechanisms governing stem cell fate, (9) regulation of energy metabolism and cell cycle progression, (10) bioenergetics of mitochondrial carrier proteins, and (11) diverse bioprocess improvement uses including clone selection for metabolic stability and optimization of media formulations.
  14. There are two reasons for incubation periods of 24 hours or more. The first and main reason is to allow time for the cells to use up all residual glucose from the serum. As we developed a general protocol for a wide range of cells using 5% serum we noticed interference from glucose in the serum. By doing a time course with a wide range of cells, we found that 40 hours was a good time period to allow the cells to use up the residual glucose in the medium and switch to using the various carbon sources in the PMM wells. The second reason is that the cells may require some period of time to "turn on" alternative non-glucose catabolic pathways.

    An important point is that one can shorten and even eliminate the 40 hour incubation if a glucose-free medium (with low glutamine) such as a serum-free medium or a medium with dialyzed serum is used.

  15. It is highly unlikely that amounts of ROS made by cells would be sufficient to interfere. We have never observed any problem, even with chemicals that interfere with mitochondrial function.
  16. Yes. Substrates and other chemical agents in the 96-well plates can be solubilized and then transferred into 96-well plates containing the adherent cells.
  17. Yes. Over the past thirty years Biolog has gained considerable experience in analyzing phenotypic assay data and is committed to helping researchers gain the most value from their use of PMM assays.
  18. PMM assays can complement and even guide the use of other approaches. PMM assays are unique in being able to provide a global assessment of metabolic activity, offering the prospect of identifying new pathways of particular importance.
  19. Mass spectrometers typically measure pool levels of metabolic intermediates. For many studies it is more important to measure pathway fluxes. Measurement of pathway flux is typically done with radioactive tracers, which is complex, expensive and requires that all pathways are known to correctly interpret the data. PMM assays provide a simple and direct measure of pathway fluxes and can elucidate unknown pathways. Furthermore, PMM assays are simple and inexpensive to set up and use, and can be performed by any laboratory.
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