McCormick Lab Wiki

This protocol will outline how to prepare your cells for fluorescent microscopy analysis with the CX7 located in the AIM core using a LIVE/DEAD assay supplied from ATT Bioquest. This protocol will also outline how to exposure your adherent cell lines to drug concentrations without worrying about media evaporation.

Before starting, ensure that the CX7 is up and running and that it can connect to the database. In the database are the procedures that Blaise has programmed into the machine to accurately assess whether a cell that is capture is alive or dead based on the fluorescence measured. This protocol uses Target Activation. For other protocols, you must coordinate with the AIM core director. Blaise will be doing LC3 quantification in MEF cells in Spring/Summer 2021 in accordance to the AFAR grand so he will outline procedures for that when he is able to.

1. Reserve the CX7 in iLab. If this is your first time doing it, plan a time for Blaise or Sharina to help out during your reserved time.
2. Split cells into the black/clear bottom 96 well plate at a density of 8000 cells/cm^2 as discussed in the subculturing procedure. Avoid seeding into the edge wells. Seeding should be done 2 days before planning to use the CX7.
3. One day after seeding, expose the cells to drugs. To do this, create media-drug suspensions at the concentration desired, using the spreadsheets provided (ask Blaise or Christine). Then, aspirate off old media and replace with this media. From now on, the aspiration of solution from your wells should be done very carefully to avoid harming your cells and messing-up your results.
4. The next day, make a working solution following the ATT BIOQUEST protocol and add that to the wells.
5. When ready to image, aspirate off the working solution and add 100ul Hank's buffer to the wells.
6. Take to CX7 and use the LIVEDEAD-ATTBIOQUEST-BLM procedure. Save the file so that you can locate it and tweak it, if needed.
7. Ensure that the machine is collecting the information that you want to get in the Population Characterization tab.
8. Scan the plate.
9. Export the data to a thumbdrive.