BSA and phenol red that interfere with the detection of this fluorescent probe should be avoided.Store aliquots at -20℃ to avoid repeated freeze-thaw.The working solution of this product should be freshly prepared before use.DAF-FM DA solidifies and sticks to the bottom of the centrifuge tube, tube wall or lid at lower temperatures such as 4℃, and can be thawed at 20-25℃ in a water bath.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.
Instructions for Use：
1. Probe loadingFor cells with a short stimulation time (less than 2 hours), load the probe first, then treat cells with an appropriate positive control and the drug of interest. For cells with a long period of stimulation (more than 6 hours), treat cells first and load the probe subsequently.In situ loading: This method is only for adherent cells. Dilute DAF-FM DA with the Dilution Buffer provided in this kit at 1:1000 to a final concentration of 5μM. Remove the cell culture medium and add an appropriate volume of diluted DAF-FM DA to cover the cells completely, usually 1ml per well of six-well plates. After incubation for 20 min in a 37℃ cell culture incubator, wash the cells three times with PBS (pH 7.4) to thoroughly remove the DAF-FM DA that did not enter cells.Load the probe after cell collection: Dilute the DAF-FM DA with the Dilution Buffer provided in this kit at 1:1000 to a final concentration of 5μM. After cell collection, resuspend cells with the diluted DAF-FM DA to 0.1-2×107 cells/ml and incubate for 20min in a 37℃ cell culture incubator. Invert the tube every 3-5 min to fully mix the probe and cells. Wash the cells three times with PBS (pH 7.4) to thoroughly remove the DAF-FM DA that did not enter cells. Treat cells directly with a positive control or drugs of interest, or aliquot cells before the treatment.2. DetectionSamples loaded with probes in situ can be observed directly by confocal microscopy, or after cell collection, examined by fluorescence spectrophotometer, fluorescence microplate reader, or flow cytometry. Samples loaded with probes after cell collection can be examined by fluorescence spectrophotometer, fluorescent microplate reader, flow cytometry, or confocal microscopy.3. Parameter settingDetect the fluorescence intensity in real-time, at each time point or at a single time point before and after cell treatment at the Ex/Em of 495/515nm. Parameter settings for detecting fluorescein or FITC can also be used.4. Other instructionsThe recommended working concentration of DAF-FM DA is 5μM. For some cells, if the negative control cells without drug treatment also exhibit strong fluorescence, the DAF-FM DA can be diluted at 1:2000-1:5000 to 1-2.5μM. Conversely, if the fluorescence is very weak after drug treatment, the working concentration of DAF-FM DA should be increased to 10μM to improve the labeling. Additionally, the probe loading time can be adjusted within 15-60 minutes.
