After incubating Crohn's disease patient-derived colonoids for three days, tilt the plate and remove the medium from the edge of the wells. Add 200 microliters per well of cytokine treatment media containing 2.5 micromolar fluorescent cell death dye. Incubate the colonoids at 37 degrees Celsius and 5%carbon dioxide for the required treatment time.
After incubation, transfer the plate to the stage of a digital-inverted epifluorescence microscope and confirm that the max toxicity condition colonoids are fully lysed. Using the transmission channel, focus on a colonoid with SYTOX-positive cells. Then, switch to the GFP channel.
Adjust the light intensity and exposure time to maximize the fluorescent signal while minimizing the background. Using optimized image settings, observe the no dye and max toxicity conditions to ensure the samples are not over or underexposed. Acquire transmission and GFP images of colonoids using a random-sampling approach by selecting fields of view that follow a fixed grid pattern covering the colonoid dome.
Save images in portable network graphic format and export them. Drag and drop the image dataset files onto the ImageJ toolbar and sequentially click Image, Stacks, and Images to Stack to combine the files. Then, click Image followed by Type and 8-bit to convert the image stack to an 8-bit file.
Click the Freehand Selections tool and manually select the region of interest on the transmission image. Then, toggle through the image stack to the corresponding GFP channel image. From the toolbar, click Analyze and Set Measurements.
In the Set Measurements dialogue window, tick the Mean gray value and leave other boxes unticked. With the GFP image selected, click Analyze and Measure. Once the dataset is analyzed, copy all the data in the results window and paste them into a spreadsheet.
Calculate the mean of the technical replicate mean gray values for each condition. Subtract the mean of the untreated condition from each treatment condition. Then, divide each treatment condition by the background-subtracted mean of the max toxicity condition.
To calculate cell viability after treatment, subtract the normalized values from one. Using the given equation, calculate the coefficient of perturbagen interaction. Transmission fluorescent overlay images of colonoids at eight hours indicated that only the interferon gamma plus TNF alpha-treated colonoids were positive for fluorescent signal.
At 24 hours, colonoids treated with interferon gamma plus TNF alpha displayed large regions positive for fluorescent signal with a clear breakdown in colonoid morphology. Cell death levels at eight hours were low for BSA-control colonoids with a slight increase in TNF alpha-treated conditions. At 24 hours, interferon gamma plus TNF alpha-treated colonoids showed the highest cell death levels.
CPI values indicated slight synergism at eight hours and substantial synergism at 24 hours. This confirmed a time-dependent synergistic interaction between interferon gamma and TNF alpha.
