Cell Counter

At the Cell Counter: PC-12 Cells

First cultured by Greene and Tischler in 1976, PC-12 cells originated from a pheochromocytoma (neuroendocrine tumor) of the rat adrenal medulla. It was developed as a model cell line and an alternative to adrenal chromaffin primary cell cultures. PC-12 cells are able to differentiate into neuron-like cells in the presence of nerve growth factor or dexamethasone. Due to their differentiation ability and ease of culture, PC-12 cells are used in a variety of research areas ranging from drug efficacy to neurosecretion.

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Figure 1. StainFree technology compared to fluorescence cell counting

PC-12 cells counted using StainFree™ Cell Detection Technology (blue circles) and red nuclear stain (red circles). Counts obtained from both methods agree closely, demonstrating that StainFree technology gives accurate cell counts while eliminating the need for fluorescent dyes (r2 > 0.99 for each plot).

Figure 2. Growth curves obtained using StainFree covered area analysis

Growth curves of PC-12 cells over a ten-day period. Cells were originally seeded at 2000 cells (red), 1000 cells (green), 500 cells (blue), and 200 cells (purple). Cell confluence was measured every 24 hours using the Field Analysis feature in SoftMax Pro Software. The observed dip in confluence for the 2000-cell initial density population at 144 hours (*) is likely due to depletion of nutrients in the culture medium; medium was originally replaced every three days, but from 144 hours onward it was replaced daily.

Figure 3. StainFree analysis of PC-12 cells with mitochondrial staining

Top row shows transmitted light images overlaid with red fluorescent (mitochondrial) images. Bottom row shows StainFree analysis with individual cells (purple masks) identified by the software. Left, untreated cells; right, cells treated with 1 µM valinomycin. For each individual cell identified, the intensity of mitochondrial staining could be calculated.

Figure 4. IC50 curves for PC-12 cells treated with valinomycin

PC-12 cells were treated with valinomycin and assayed for mitochondrial activity using MitoTracker™ Deep Red FM (MTDR) dye. Concentration-dependent response curves are shown for cells analyzed using StainFree Technology (blue) or fluorescent nuclear staining (green) to identify individual cells. Average mitochondrial fluorescence per cell is graphed vs. concentration of valinomycin. Nearly identical curves were obtained with both analysis methods, and in both cases the IC50 value was 57 nM.

Tip 1:

To count PC-12 cells without staining, we recommend creating a new setting using the drawing tools in the software. Choose the Discrete Object Analysis and then use the drawing tools to define cells and non-cell areas in your max and min images. To get more accurate counts in dense clusters of cells, try drawing just within the borders of the cells. Conversely, if you find that the software is over counting the cells in your images, try drawing slightly outside the borders of the cells.

Tip 2:

It’s easy to get growth curves with the Field Analysis feature in SoftMax Pro Software. Field Analysis calculates the percent area covered by cells (confluence) in your images. Measuring cell confluence can be a valuable tool for better assay development. For example, you may seed cells at two or more test densities, calculate the confluence of your cells just prior to assay, and upon obtaining assay results, determine what level of confluence yielded the best results.

PC-12 Cells Analysis Toolkit

Instrument Settings

Setting for cell counts
Setting for growth curves
Optical configuration
SpectraMax MiniMax 300 Imaging Cytometer
Read mode
Read type
Wavelength settings
Transmitted light
Image acquisition settings
Exposure: 5 ms
Exposure: 7 ms
Image analysis settings
Analysis type: Discrete Object Analysis Wavelength for finding objects: TL
Analysis type: Field Analysis Wavelength for finding objects: TL
Find objects
Predefined setting: CellsA
For PC-12 cultures, a user-defined setting may give the best results. Image acquisition and analysis settings may vary depending on assay conditions, microplate used, and other factors.

About StainFree Cell Detection Technology

Imaging cell-based assays typically requires the use of fluorescent probes that can be toxic to living cells or may only function in fixed cells. A label-free method for analyzing cell counts and cell confluence enables researchers to quantitatively monitor cell proliferation and health without time-consuming workflows that may disrupt cell viability.

The SpectraMax i3 Multi-Mode Microplate Platform with MiniMax 300 Imaging Cytometer uses unique, patent-pending StainFree Cell Detection Technology that allows you to perform cell proliferation, cytotoxicity, and other assays without nuclear stains like DAPI, which intercalates with DNA, or live cell dyes that are actually toxic to cells in the long term.