Logos LUNA-FL, Руководство пользователя

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6.5 Trouble Shooting
6.5.1 (Problem 1) The FL signal is too weak.
If your sample was grown in the YPD medium, be sure to dilute the sample at least 1:100 with Cell
Dilution Buffer. The component of the YPD medium has a strong esterase activity and inhibits the uptake
of FDA into the yeast cells, leading to weak fluorescence signal. For the detailed information about the
effect of YPD, please read our latest application note (www.logosbio.com).
6.5.2 (Problem 2) The FL signal is still weak so that some yeast cells were not counted.
→ Increase the exposure up to 20.
6.5.3 (Problem 3) The FL signal is present, but it was not counted (circled).
→ Decrease the threshold down to 1.
6.5.4 (Problem 4) The background is still bright even after the dilution or washing step.
→ The Fluorescence Signal Enhancer 1 (Cat # F23213) can be used. See the following protocol.
<Using the Fluorescence Signal Enhancer 1 (Cat # F23213)>
1. Dilute yeast cells up to 1/100 with the LUNA-
FL™ dilution buffer. Or, centrifuge the
yeast culture and resuspend in the LUNA-
FL™ dilution buffer.
2. Mix 1 µl of the Fluorescence Signal Enhancer 1 and 17 µl of the yeast sample.
3. Incubate for 10 min.
4. Add 1 µl of FDA and 1 µl of PI.
5. Incubate for 10 min.
6. Load 10 µl of the stained yeast sample on the counting slide.
7. Wait for 30 sec-1 min or until all yeast cells are immobile.
8. Touch the
“Count” button.
Note : Remember that the Fluorescence Signal Enhancer 1 will be helpful, depending on the sample
conditions. For example, it is helpful for exponentially growing yeasts, but, sometimes not for end-of-
culture yeasts. The rapidly dividing yeasts are metabolically active and they try to pump out the activated
fluorescein. The enhancers are actually inhibitors against the pumping/transporting mechanism. When
used in proper conditions, the enhancers will lower background and raise the intensity of signal, thereby
enhancing overall signal to noise ratio.