The protocol describes a rapid, high-throughput, reliable, inexpensive, and unbiased assay for efficiently determining cellular viability. This assay is particularly useful when cells' mitochondria have been damaged, which interferes with other assays. The assay uses automated counting of cells stained with two nuclear dyes – Hoechst 33342 and propidium iodide.
This method will allow researchers to assess cellular viability independently of mitochondrial function. And it is amenable to high throughput drug screening efforts. This technique is fast, accurate, inexpensive, and allows the determination of compounds cytotoxicity, including those impairing mitochondrial function in most cells types.
This protocol is easy to perform. It is important to pay attention to the drug preparation, treatment conditions, and cell density to ensure experimental accuracy and validity. Visual demonstration of this method is important because image acquisition and image analysis steps can be hard to recapitulate, especially for researchers having little experience with Gen5 Software.
Begin by preparing solutions of the compounds of interest at the desired concentration. Always make sure to mix compounds by vortexing thoroughly. To measure cytotoxicity of a single compound, prepare compounds at 2X final concentration.
If measuring cytotoxicity of compound combinations, prepare them at a 4X final concentration. Prepare solvent only controls by mixing the same amount of solvent with the appropriate medium. Collect cells into a 15 milliliter conical tube and aliquot 10 microliters of the cell suspension.
For trypan blue staining, add 10 microliters of 0.4%trypan blue to the aliquot and use a hemocytometer or cell counter to count viable and non-viable cells. Pellet cells in the 15 milliliter tube at 200 times G for five minutes, then aspirate or decamped the supernatant. We suspend the cell pellet in essay appropriate media at a cell density of three times 10 to the fifth cells per milliliter.
Use a multi-channel pipette to seed 50 microliters of cell suspension into each well of a 96 well plate. For single compound assays, add 50 microliters of 2X compound solution into each well. For solvent control wells, add 15 microliters of test media containing the solvent at 2X concentration.
For combination assays add 25 microliters of each of the 4X compounds into each well. For single compound control wells, add 25 microliters each of 4X compound solution and test medium. Make sure that the final concentration of DMSO does not exceed 0.5%To ensure the reproducibility, add drugs with the pipette tips touching the side of wells.
Being careful to add the drugs to different locations. This stabs should be performed very quickly, preferably taking no longer than 30 minutes per plate. Gently tap the plate to mix the contents of the wells and incubate it at 37 degrees Celsius and 5%carbon dioxide.
When ready to stain the cells with Hoechst 33342 and propidium iodide prepare fresh 10X staining solution and add 10 microliters to each well. Gently tap the plate and incubate it at 37 degrees Celsius for 15 minutes. Centrifuge the plate at 200 times G for four minutes to bring all the cells to the bottom of the plate.
Then carefully wipe the bottom of the plate with a damp laboratory wipe to remove any debris that may interfere with imaging. Image the plate within 15 minutes after centrifugation. Open Gen5 Software and create a new standard protocol, click on Procedure and choose the type of plates to be used.
Usually, 96 well black plastic plates. Next, set the read method to image, click Okay, and choose DAPI and Texas Red Filter sets with a 4X magnification objective. No offset is needed, since the well centers will be imaged.
For the DAPI filter, set LED to 10, integration time to 99, and gain to zero. For Texas Red, set LED to eight, integration time to 950, and gain to 18. Click on Options to make sure that the auto focus is performed on the DAPI channel and there is no offset in focusing between channels.
Save the protocol by clicking Okay. Now images can be recorded using the Read New button. Once images are recorded, click on Data Reduction and choose Image Preprocessing.
Apply Image Preprocessing with dark background subtraction using Auto flattening size based on the DAPI signal. For Texas Red, use the same options as for Channel 1. When finished, click Okay.
Then go to the cellular analysis panel and apply a Nuclear Mask based on the transformed DAPI signal with a threshold value of 6, 000 units, minimal object size of five micrometers and maximal object size of 25 micrometers. Analyze the entire image, exclude primary edge objects on the border of the image and split touching objects. In advanced detection options, set the rolling ball diameter as 30 micrometers, and evaluate the background based on 5%of lowest pixels.
Only keep the cell count in calculated metrics. After that, perform a subpopulation analysis based on the mean transformed Texas Red signal with a threshold value of 5, 000 units to count dead cells. Finally, access the resulting cell counts.
Representative images of cells stained with Hoechst, propidium iodide are shown here. The total number of cells is reasonably high and greater than the number of dead cells. When a panel of cytotoxicity assays was compared with trypan blue exclusion, it was found that MTT and the Alamar blue assays inaccurately measured cellular viability.
These mitochondrial enzyme-based assays showed significantly lower viability compared to trypan blue exclusion. Dual staining with Hoechst 33342 and PI had the best combination of robustness, sensitivity, and consistency with trypan blue staining and the smallest median deviation from the trypan blue exclusion method after CCP or 2-DG treatment. The Hoechst PI essay was also effective at determining cellular viability after treatment with the mitochondria targeting molecules Rotenone and three bromopyruvate.
It was further validated with a panel of leukemia cell lines. These cells varied in Rotenone sensitivity ranging from very sensitive to resistance cells. Improper performance of the assay may compromise its accuracy.
Several compromised outcomes are shown here. Overstaining with PI, neglecting the centrifugation step, or overexposure in the Hoechst channel, will cause suboptimal results. While timing this protocol, remember to optimize the dye concentration and staining time for each cell line.
Also, centrification for a sample plate, is necessary to ensure imaging quality. After identification of compounds of cytotoxicity effect on cancer cells, researchers may proceed with mechanistic studies to identify their relevant targets. Screening small molecule libraries with this technique in parallel to conventional MTT assays, will allow molecules that target mitochondrial function to be rapidly identified.
