Why Software?
Throughout the course of this project, we found that we had spent a great deal of time repeating calculations for transfection, light conversions, and mammalian culturing and passaging. To lessen this computational burden, we created calculators which input raw lab data and output useful values. We hope that these calculators will aid other iGEM teams who are working with mammalian cells or light based systems by reducing the time spent on calculations.
Check out our Calculator for Passaging Mammalian Cells:
Our Mammalian Passaging Calculator is intended to aid teams working with mammalian cells. It can be used to calculate the total number of cells in a sample based on hemocytometer counts. It can also calculate the volume of cells to use when seeding plates and T75 or T25 flasks. Additionally it can be used to make freezer stocks.
To use the Mammalian Passaging calculator, first passage the mammalian cells according to our Mammalian Passaging Protocol, and resuspend in media. Using a haemocytometer and TryphanBlue, count your cells. Input the cell counts for each quadrant of the haemocytometer in the Calculator, then select the number of cells you would like to seed in each well or T75, then select the desired number of T75’s, plates, or freezer stocks.
Try Our Transfection Calculator
For mammalian transfection, we created an easy to fill in google sheet for adjustable plasmid ratios, DNA concentrations, lipofectamine concentrations, and number of wells. To use it, download this spreadsheet and fill in your values!
Cell Counting for Fluorescent Imaging
ImageJ Cell Counter
Download Fiji/ImageJ for your device.
To reduce time spent and human error in cell counting, we developed automated cell counters on ImageJ/FIJI. These simple to use “Macros” streamline transfection efficiency measurements. Cell count, and consequently transfection efficiency, can be estimated through analyzing both the transmitted Bright field and GFP images, and then taking the ratio - GFP cells:transmitted cells. Contrast, threshold, and color balance can be corrected within Fiji. We found that the type of processing required varies depending on the contrast of raw images (high or low) and the amount of fluorescence (high or low). In order to run the same program with multiple pictures, we first created a macro for each category. We created 4 programs for the different conditions. Transmitted cell pictures can be processed using either BF_cell_count_high _contrast.ijm or BF_cell_count_low_contrast_ijm, and GFP pictures can be processed using brightFluorescence.ijm or dimFluorescence.ijm.
To begin making a macro, open up a picture in Fiji. Go to Plugins > Macros > Record. The program will automatically generate code that can be saved for future use as you click on the items in the top menu. An alternative shortcut is typing the last command ( menu selections as noted in the scripts) in the search bar under Help. For more information about using ImageJ or FIJI for automated cell counting, please read our Fluorescence Imaging and Flow Cytometry Protocols on Adherent Mammalian Cell Lines Protocol. To download a Macro, click on the cell condition that you would like to analyze.
Check out our Conversion for Light Intensity:
To compare our results to literature and to other iGEM teams, we created a simple converter between common units of light intensity.
