Nexcelom Bioscience

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Using Cellometer Image Cytometry to Detect and Measure GFP Expression Efficiency

Video Category: Cellometer Vision CBA Image Cytometer

This presentation will focus on using Cellometer image cytometry to effectively utilized to determine GFP or other fluorescent protein transduction/transfection efficiency and cell concentration as well as detect and quantify dual expression in a cell population.

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Transcript: Using Cellometer Image Cytometry to Detect and Measure GFP Expression Efficiency

  • Welcome to the Nexcelom Bioscience webinar. The topic for today's discussion is using Cellometer image cytometry to detect and measure GFP expression efficiency.
  • GFP is a green fluorescent protein that is 26.9 kDa in size. It was first identified in crystal jellyfish Aequorea Victoria and when exposed to blue or ultraviolet light the GFP fluoresces green. GFP was first expressed in E.coli in 1994, and has since been successfully incorporated and expressed in a great variety of bacterial and mammalian organisms. Many fluorescent proteins of different colors have been generated and their function has been enhanced to provide faster and stronger fluorescent signal.
  • Quantifying GFP can be done in four easy steps. First pipette 20 ul of cell sample into a disposable counting chamber. Then insert the slide into the instrument, select your assay from the drop down menu, and click count. Within seconds the instrument acquires images, identifies cells with and without fluorescence and automatically tabulates the results. The results include the number of cells counted in bright field and those that are GFP positive, the concentration for each population as well as their mean diameter. Also reported is the percent of GFP positive cells.
  • Green fluorescent protein has multiple applications in cellular and molecular biology. In this case GFP or RFP is used as a reporter of gene or protein expression. By detecting GFP expression, it is possible to quantify the transfection or transduction efficiency in a population of cells.
  • Here we are looking at the GFP expression efficiency for a lentiviral transduction in Hek293 cells. Three data points were collected for this experiment: sample 1, sample 2 and control. The micrographs for each sample show a bright-field image and a corresponding fluorescent image. The captured images were analyzed and the results are displayed below. In Sample 1, 58% of the cells are GFP positive. When the lentiviral vector is diluted two-fold the percent of GFP positive cells decreases from 58%- 24.1%. The control sample does not contain a vector and therefore no GFP expression was detected.
  • Similar to GFP expression, we can also detect RFP expression
  • Shown here is RFP expression in RWPE-1 cells. Bright-field and fluorescent images were acquired and the data output table shows the number of cells counted, the concentration of bright-field cells and RFP positive cells, the mean diameter and the percent of RFP positive cells.
  • It is also possible to examine GFP expression and cell viability in a single assay. This is accomplished by staining the cell sample with propidium iodide.
  • In this example, GFP-expressing mouse embryonic stem cells have been stained with propidium iodide. Bright field, GFP and PI images were captured and analyzed. The results include counts for GFP positive cells, PI positive cells, the total number of cells in bright field, as well as the concentration for each population, and the mean diameter. Also reported is the percent of cells that are GFP positive, the percent of cells that are PI positive, and the population of cells that are dual positive. Since only 0.1% of cells are both GFP and PI positive we can conclude that nearly all of the cells, greater than 99%, that are expressing GFP are viable cells.
  • The third application examines cells in a culture that co-express GFP and RFP. Cellometer can capture, analyze and report the percent of cells that are GFP positive, RFP positive or dual positive.
  • In this example we are looking at T-cells that are co-expressing GFP and RFP. Bright field, GFP and RFP images are acquired and analyzed. The reported results include the number of cells counted for each population, the concentration, the mean diameter and the ratios of GFP positive, RFP positive and dual positive cell populations. In this example about 38% of the cells are both GFP and RFP positive.
  • Alternatively we can also measure GFP expression by looking at the total fluorescent intensity of a sample and export it to software typically used for flow cytometry. The data acquired from the Cellometer images are exported to FCS express 4 software and a histogram is generated. The histogram displays the GFP negative as well as GFP positive populations. Gating can be manually adjusted and the corresponding data is automatically updated. The data table contains the percent of GFP positive and GFP negative cells and their corresponding concentrations.
  • Our customers have successfully detected and quantified GFP expression in many different mammalian cell lines. Represented here are just some examples of those GFP expression experiments. A bright-field and a GFP micrograph is shown for each GFP expressing cell-line: COS 7, 293F, HeLa cells. As well as, T3T, H1299, and K562 cells.
  • Detection of GFP expression can also be performed in yeast. These micrographs show GFP-expressing Saccharomyces cerevisiae. Since the imaging of yeast requires a higher magnification, these particular Bright-field and fluorescent images were captured by a Vision10x instrument.
  • Not only can the Cellometer detect GFP and RFP expression, but it can also detect and analyze any fluorescent proteins, within the UV to far red spectrum.
  • The Cellometer interchangeable fluorescent optics modules are optimized for detection of various fluorescent signals. These fluorescent optic modules are not only used to measure fluorescent proteins but they are also capable of measuring enzymatic dyes, AlexaFluor dyes, and DNA-binding dyes.
  • In conclusion, Cellometer image cytometry may be effectively utilized to determine GFP or other fluorescent proteins transduction or transfection efficiency, calculate cell concentrations, as well as detect and quantify dual expression in a cell population such as GFP/RFP. Single assay analysis may be performed to simultaneously measure GFP expression and cell viability by staining the cells with propidium iodide. It may also be used to study not only GFP expression in mammalian cell lines but also in GFP expressing yeast cells by using our Vision10x instrument. This robust instrument platform along with separate software- solutions provides researchers with the ability to detect and directly compare fluorescent signals.
  • Detecting and measuring GFP expression is a common assay in many research fields. Our customers have published in a variety of journals from DNA Repair to Journal of Virology to Molecular Cancer Research. Thank you for your time.