Green Fluorescent Cathepsin B Assay

1. Prepare samples and controls., 2. Dilute 10X Cellular Assay Buffer 1:10 with diH2O., 3. Reconstitute Calcein AM with 50 µL DMSO to prepare 2 mM stock solution., 4. Dilute 2 mM Calcein AM stock solution 1:5 by adding 200 µL diH2O or PBS, forming a 400 µM stock solution., 5. Stain with Calcein AM at a concentration between 1-10 µM. The ideal staining concentration can vary based on cell line, application, etc., and should be determined by the end user. The recommended sample size is 0.4 mL., 6. Add Calcein AM to each sample and mix gently. For example, to stain a 0.4 mL sized sample with 10 µM Calcein AM, add 10 µL of the 400 µM stock solution., 7. Incubate approximately 1 hour., 8. Analzye with a flow cytometer, fluorescence microscope, or fluorescence plate reader. Calcein AM excites at 494 nm and emits at 520 nm.