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The the EFLUXX-ID® multidrug resistance assay kits (ENZ-51029, ENZ-51030) have been validated for Flow Cytometry and Microscopy. This does not necessarily mean that they are not compatible with analysis on a microplate reader properly equipped. Just that we cannot guarantee for the results obtained with this application. Please see below some general indication that might help in setting up the protocol for an experiment condacted on a microplate reader. Optimization ight be required: 1. Seed cells in black 96-well plates (1×105 cells/well, 8 replicates for each treatment condition). For each set of experiments, prepare an empty raw of wells.
2. The following day, remove the seeding medium and treat the cells at 37°C for 15 minutes with the following inhibitors (provided with the kit) in indicator-free growth medium (use the indications in pages 10 of the manual for further instructions on inhibitor preparation, and adapt to volumes to your needs):
a. Verapamil, final concentration: 20 μM
b. MK-571, final concentration: 50 μM
c. Novobiocin, final concentration: 100 μM
d. Vehicle (DMSO).
3. Add eFluxx-ID ™ Green probe (200 times diluted in final concentration; use indicator-free medium) and stain the cells with the probes with or without inhibitors for additional 30 minutes at 37°C (use the indications in pages 10 of the manual for further instructions on EFLUX-ID preparation, and adapt to volumes to your needs).
4. Measure the fluorescence either immediately or remove the excess of dye by washing with PBS (wash with 200μL of ice-cold PBS, add 100μL of fresh ice-cold PBS and read)
5. To calculate MDR, all data should be normalized by subtracting empty well background. Fluorescence ratios for inhibitor-treated vs untreated cells should be calculated.