Jurkat cells were used to analyze cell cycle kinetics following treatment with the cell-cycle-arresting drug etoposide.  Etoposide is designed to arrest the cells at the G₂ phase of the cell cycle. Jurkat cells were incubated with media only (control) or etoposide (0.06 µM, 0.12 µM) for 24 hours. Control and drug-treated cells were ethanol fixed and stained with cell cycle propidium iodide reagent. For each sample, 20 µl of cell sample (at ~4 x 10⁶ cells / mL) was loaded into a Cellometer imaging chamber, inserted into the Vision CBA Analysis System, and imaged in both bright field and fluorescence. The fluorescence intensity for each cell was measured and a cell cycle histogram is automatically generated for each sample using the optimized Nexcelom cell cycle data layout in FCS Express 4 Flow Software. Gating can be manually optimized directly on the histogram with automatic update to the associated data table.

Figure 1. Control Population histogram for Jurkat control sample generated with FCS Express 4 software. As expected, most cells in this control sample are in the G0/G1 phase of the cell cycle. Representative data for this histogram is can be seen in Table 1.

Table 1. Control Data Population distribution of Jurkat cells at different cell cycle phases. Nearly 55% of the gated cells are in the G0/G1 phase and are therefore, either resting or preparing to begin the next round of cell division. Approximately 15% of the gated cells have completed DNA replication and are preparing to enter the mitosis phase.

Figure 2. 0.06 µM Etoposide Population histogram for the etoposide (0.06 µM) treated Jurkat cells. There is a noticeable increase in the percent of cells that have arrested in the G2/M phase of the cell cycle (see Table 2). This can be seen in the increase in the G2/M peak (green gate) at ~ 4200 relative fluorescent units (RFUs).

Figure 3. 0.12 µM Etoposide Population histogram for the etoposide (0.12 µM) treated Jurkat cells. At this drug concentration, most of the cells have now arrested at the G2/M phase. On the histogram, this can be seen as shift in the cell population from G0/G1 (blue gate) to G2/M (green gate) when compared to control and 0.06 µM etoposide samples.

Table 2 shows the population distribution for control and etoposide treated cells. The treatment of Jurkat cells with etoposide arrested the cells in the G2/M phase of the cell cycle. As the etoposide concentration increased, the percent of cells that arrested in the G2/M phase of the cell cycle also increased from 15.4% to 52.2%.

The Cellometer fluorescence-based cell cycle analysis can be effectively implemented in studies examining the efficacy of cell cycle arresting drugs.

Introduction

One of the most common and popular methods for cell cycle detection is the use of fluorescence-based dyes. There are a number of fluorescent-based dyes that are capable of binding to double stranded DNA upon cell fixation. Propidium iodide (PI) and DAPI are two such dyes. Since the amount of bound fluorescent dye is directly proportional to the amount of DNA present within a cell, these dyes can be used to detect the cell cycle within a population of cells.

How it works:

As the cells are going through the cell cycle, the amount of DNA that is contained within the cells is dependent on which phase of the cell cycle the cells are in.

Since the amount of DNA doubles from 2n to 4n between G1 and G2/M phases, and the amount of PI incorporated is correlated to the amount of DNA within each cell, we can generate a histogram based on PI fluorescence intensity.

The Cellometer instrument acquires a bright-field and a fluorescent image for each sample tested. The bright-field image allows researchers to verify cell morphology, evaluate the degree of homogeneity of the sample, and identify the presence of cellular debris

The fluorescent counted image can be used to confirm that cells are counted correctly. Individual counted cells are outlined in green. Uncounted cells are outlined in yellow. Cellometer software uses proprietary algorithms to accurately count individual cells within clumps.

Once counting is complete, a cell cycle histogram is automatically generated for each sample using the optimized Nexcelom cell cycle data layout in FCS Express 4 Flow Software. Gating can be manually optimized directly on the histogram with automatic update to the associated data table.

Representative data set:

In this example, untreated Jurkat cells were used to analyze cell cycle kinetics. The cells were ethanol fixed, stained with propidium iodide cell cycle reagent, and 20 µl of cell sample was loaded into a Cellometer counting chamber.  The counting chamber was inserted into the Vision CBA Analysis System, and imaged in both bright field and fluorescence. The fluorescence intensity histogram and associated data table was generated in FCS Express 4 software.

Figure 1. Control Population histogram for control sample generated with FCS Express 4 software. Sub-G1 population is in red, G0/G1 population is indicated in blue, S population is in brown, and G2/M population is in green. Each phase gate can be manually adjusted for each sample.

Table 1. An automatic data table is generated. The table contains the percent of gated cells at each cell cycle phase as well as cell concentration.

Calcein AM (Calcein acetoxymethyl ester) is a non-fluorescent compound that passively enters cells. In metabolically active cells, Calcein AM is converted by cytosolic esterases into green fluorescent Calcein. The fluorescent Calcein is retained by live cells with intact membranes. Only cells possessing active cytosolic esterases fluoresce green. This allows for quick and easy detection of metabolically-active (vital) cells in a sample. (more…)

GFP and Determination of Transfection Efficiency

GFP positive cells imaged with Cellometer Vision CBA

Green Fluorescent Protein (GFP) is a 26.9 kDa protein that fluoresces bright green when exposed to blue or ultraviolet light. GFP was first identified as a protein and extracted from the Aequorea victoria  jellyfish in 1962 by Osamu Shimomura, et al.  The GFP protein was first cloned in 1992 and it was soon confirmed that GFP protein expressed in other organisms generates fluorescence. An area within a cell or tissue is briefly illuminated, causing the GFP protein to fluoresce, allowing GFP-tagged proteins to be identified. In 2008, Osamu Shimomura, Marty Chalfie, and Roger Tsien received the Chemistry Nobel Prize “for the discovery and development of the green fluorescent protein.” In recent years, various mutations have yielded forms of GFP offering improved brightness, a modified excitation and emission spectra, improved 37°C folding, reduced aggregation at high concentrations, and improved diffusibility inside cells. Today, GFPs are used in an increasing variety of cell-based assays to measure intra-cellular events. GFP genes are co-transfected with genes of interest to measure transfection efficiency, GFP fusion tags are used to identify the localization and fate of normal host proteins, and specifically-engineered GFPs are used as indicators of phosphorylation.

With the Cellometer Vision Image Cytometry Systems, researchers can easily quantify the percentage of GFP-positive cells within a population, allowing for fast, accurate determination of transfection efficiency. Researchers can overlay histograms for control and treated samples for advanced gating of GFP- and GFP+ cell populations.

¹Tsien, Roger Y., (1998). The Green Fluorescent Protein, Annu. Rev. Biochem. 67:509-44.

²http://www.conncoll.edu/ccacad/zimmer/GFP-ww/timeline.html,accessed September 25, 2012.