We tested the following cell lines with this kit.

We have tested fixing cells after staining with 4% PFA.

〈Protocol〉

－Adherent cell－
1. Prepared the cell in a dish or microtube.
2. Removed the medium, and washed the cells twice with serum-free medium.
3. Added serum-free medium, incubated at 37℃, 5% CO₂ for 15 min.
4. Removed the medium, Added Fatty Acid Uptake Probe working solution, then incubated at 37℃, 5% CO₂ for 15 min.
5. Removed the medium, and washed the cells once with Washing Buffer solution.
6. Added 4% PFA/PBS to the cells, incubated 5 min at room temperature.
7. Washed the cells 3 times with PBS, and then tested the samples with a fluorescence microscope and a plate reader.

*Please be aware that cell fixation may decrease the fluorescence intensity.

－Floating cell－

1. Prepared the cells in a microtube.
2. Centrifuged at 300×g for 5 min and then removed the supernatant.
3. Added serum-free medium, after suspending the cells by pipetting, centrifuged at 300×g for 5 min, and then removed the supernatant. Repeated this step once.
4. Added serum-free medium, after suspending the cells by pipetting, incubated at 37℃, 5% CO₂ for 15 min.
5. Centrifuged at 300×g for 5 min, and then removed the supernatant.
6. Added Fatty Acid Uptake Probe working solution, after suspending the cells by pipetting, incubated at 37℃, 5% CO₂ for 15 min.
7. Centrifuged at 300×g for 5 min, and then removed the supernatant.
8. Washed the cells once with the Washing Buffer solution.
9. Added 4% PFA/PBS, after suspending the cells by pipetting, incubated 5 min at room temperature.
10. Centrifuged at 300×g for 5 mins, and then removed the supernatant.
11. Added PBS, after suspending the cells by pipetting, centrifuged at 300×g for 5 min, and then removed the supernatant. Repeated this step twice.
12. Tested the samples with a fluorescence microscope and a flow cytometer.

Please use a black microplate for fluorescence assay.

*The cell number needs to be sufficient to cover the bottom of the well, after the cells settled on the bottom of the well, it is possible to assay.〈Floating Cells Sample using Quenching Buffer Procudure〉

We have also tested with the following microplates.

The Fatty Acid Uptake Probe working solution can't be stored for a long period of time.

One of the possible reasons is that there was some of the Fatty Acid Uptake Probe still remaining in the well, please wash the cells one more time with the Washing Buffer solution or consider using the Quenching Buffer procedure.

Please refer to the following table.

When using a confocal microscope, transmitted light mode with 488 nm is not possible. When performing transmitted light observation, please dilute the Quenching Buffer 10-fold with Washing Buffer or using a 640 nm laser.

〈Image of the transmitted light mode of confocal microscope〉

This kit can't quantify fatty acid.

You can't quantify the uptaken Fatty Acid Uptake Probe. This kit is for fatty acid uptake ability evaluation.

The Fatty Acid Uptake Probe shows a slight leakage to red fluorescence. Therefore, please use dyes other than green and red fluorescence for co-staining.

We have tested using mitochondrial fluorescence probe MitoBright LT Deep Red (MT12) for co-staining.

〈Red fluorescence leakage〉

〈Protocol of MitoBright LT Deep Red co-staining〉
1. Prepared the cells in a dish or microtube.
2. Removed the medium, and then washed the cells twice with serum-free medium.
3. Added serum-free medium, incubated at37℃, 5%CO₂ for 15 min.
4. Removed the supernatant, added Fatty Acid Uptake Probe working solution, and then incubated at 37℃, 5%CO₂ for 15 min.
5. Removed the supernatant, added 0.1 µmol/l MitoBright LT working solution, and then incubated at 37℃, 5%CO₂ for 30 min.
6. Removed the supernatant, and washed the cells twice with HBSS.
7. Added HBSS, and then observed with a fluorescence microscope.

During fluorescence imaging, continuous exposure to excitation light may cause photobleaching of the Fatty Acid Uptake Probe. Please refrain from continuous irradiation.