FAQ

FAO Blue (Fatty Acid Oxidation Detection Reagent)

FAO Blue Fluorescent Dye Compatibility with Fluorescent Plate Readers

Yes, FAO Blue fluorescent dye is fully compatible with fluorescent plate readers for quantification purposes after cellular staining. Our log book showed that a few of our customers are using it for Flow Cytometry and a Plate Reader for quantification. We want you to know the original purpose for the dye is for a 'live cell imaging'. However, some scientists are tweaking protocols for their own use. FAO Blue (Fatty Acid Oxidation Detection Reagent) can be effectively measured using fluorescent plate readers with appropriate filter settings, so please make sure your reader has the correct filters. The dye is specifically designed as a metabolic indicator that shows blue fluorescence (emission at 430-480 nm) when excited at 405 nm after being processed through fatty acid β-oxidation (FAO) pathways in living cells. We assume that you plan to grow cells in wells (i.e.,96 well plate) and read the fluorescent output on the reader.

• We recommend Specific Plate Reader Parameters
• Excitation wavelength: 420±5 nm
• Emission wavelength: 460±10 nm
• Measurement mode: Transparent mode

This configuration allows for accurate quantification of FAO activity in various cell types in a high-throughput format. So, you may need to optimize the growing conditions and fluorescence by using microscope first before you optimize on well plates.

Recommended Protocol for Plate Reader Measurement (you may need to tweak this more to fit your cell type and experimental conditions => more optimization may be needed)

• Seed cells in 96-well plates and culture for 1 day; please make sure cells are growing in a log phase and not more than 70% confluent.
• Treat cells with test compounds in serum-free and phenol red-free medium (if applicable); If you need phenol-free or best cell culture media, our sales team can help you. We have the world's cleanest culture media and buffers to minimize the background noise (this background noise often causes problems in imaging and quantification)
• Wash cells with HEPES-buffered saline (HBS); wash quickly a few times will and then move to HBS to help reduce the noise; blank the negative control measurement- only read positive signal
• Incubate cells with FAO Blue (recommended concentration: 10 μM) in HBS for 2 hours; The time of this incubation needs to be optimized for your cell type (i.e., primary cells may be very sensitive, so you may need to shorten the time)
• Measure fluorescence directly without washing using a fluorescent plate reader

Signal Normalization

When using FAO Blue with plate readers, normalization techniques can be applied for more accurate quantification. Fluorescent signals should be normalized against the background signal of FAO Blue (same concentration as used in the experiment) without cells (this is true background; you need to subtract this). This approach allows for the detection of relative changes in FAO activity across different experimental conditions.

Troubleshooting for Plate Reader Applications

• If encountering difficulty in detecting signals with a plate reader
• Establish appropriate negative controls to subtract background/autofluorescence
• Ensure cells are metabolically active (maintain cell confluency below 70%)
• Use software to amplify positive signals linearly above background noise

If you need buffers and media that are freshly made (microbatch), perfectly pH adjusted with precision and cleanest in the world, please let our sales team know your needs.

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[KOD One] Troubleshooting High-LNA Duplex PCR with KOD Master Mix: A Guided Optimization Approach

Q: The amplification challenges using your KOD master mix for a duplex containing two single-stranded DNA (ssDNA) sequences. Each ssDNA, 50 base pairs in length, includes 20 locked nucleic acid (LNA) bases.

(1) You have a tough PCR experiment with complexity: With 40% LNA content (20 out of 50 bases) in each strand of your duplex, you may need specific protocol adaptations. We have no recommendation to guarantee success but list some potential strategies to overcome PCR amplification problems using KOD Master Mix with LNA-modified templates.

(2) LNA modifications already throw a 'curveball' with significantly altered nucleic acid properties for PCR amplification, but with 40% LNA, it is tough. The primary challenges with your template include the high percentage of LNA modifications (40%), potential clustering of LNA nucleotides within the sequence, and the relatively short template length (50 bp). Although KOD DNA polymerase can efficiently catalyze the synthesis of DNA duplexes containing numerous LNA nucleotides (but 40% LNA is a tough one), especially if you have consecutive LNA sequences in the template.

(3) We assume that you use a KOD One PCR Master Mix (most recent generation of KOD).

a. Perform gradient PCR to obtain the best Tm for your template and get this part out of the way
b. After selecting one Tm that works best, try out additives to your PCR Rx mix
* using that optimal Tm, try 1%, 2%, 3% ...up to 7% DMSO and find the best combination of Tm+DMSO.
c. With the best Tm+DMSO combination, try out Ethylene Glycol at 1%, 2%, 3% ...up to 10% (these will be 10 different PCR reactions). This may give you the best Tm+DMSO+EG combination
d. With the best Tm+DMSO=EG combination, you may want to play around with Mg++ concentration in a similar fashion, but playing with Mg++ is the last optimization

PCR Rx

(I) Do try to completely denature the initial cycle with 5 min denaturation before getting repeated cycles
(ii) Do try a very short extension time, 5 seconds to 10 seconds
(iii) Do try 20, 30, and 40 cycles. Sometimes, limited cycles help
(iv) make sure your primers are HPLC purified and highly pure; make sure your primer is designed for Tm == well above >60°C == 30bp or more can help
(v) See if your template can prevent consecutive LNA side by side
(vi) make sure your template is highly pure and no dirty inhibitors/chemicals/EtOH or any source of contaminatiomn
(vii) You can try different extension temperatures sequentially = for example, first 5 cycles at 72 °C, next 5 cycles at 70 °C, then next 5 cycles at 68...etc, so on.

** You have a tough PCR rx to solve, and it will require a careful step-by-step optimization, resolving one parameter at a time. You do not want to solve problems simultaneously since it will be ineffective. Need to solve one problem at a time and only work with the optimal parameter each time.

LipiORDER [1]

LipiORDER product

LipiORDER, Product#: FNK-FDV-0041

Q: In adherent cells, we see a lot of tubes and puncta (corresponding to ER, Golgi, Lysosomes..etc.). What do you think about the rim around the cell that represents the plasma membrane part? We are using single-color green or red fluorescence.

LIVE CELL IMAGING product [1]

* The supplier does NOT provide samples for LIVE-CELL-IMAGING product. If the $cost is an issue, our sales team can compensate for the expense by giving deep discounts on consumable plastics or similar.

FAQ from customers

1. Can the dye used for Flow Cytometry?
A few of customers tried the dye for FLOW. Although the dye is designed for a live-cell-imaging, some scientists have optimized experimental settings by using mild fixative (i.e., 70% EtOH) and very short fixation time (i.e., <30 seconds), and the experimental data was satisfactory. We do not guarantee the dye will work for all cell types and experiments. In all cases, researchers must optimize the steps of protocols for their own cell types and experiments.

2. Does the dye work for frozen tissues and fixed tissue sections?
Although the dye is for a live-cell-imaging, some researchers optimized methods to gain the data they want. One common optimization shared verbally by customers is that they started with the live-cell imaging to optimize the incubation time for their cell type of interest. Afterward, researchers performed the time course experiment with the fixative of choice and found the optimal incubation time. Some customers have used tissue and the conclusion was that each research group had to optimize conditions for their own tissues of interest (i.e., tough versus soft tissues & frozen versus FFPE.. etc.).

3. What concentration is recommended for cell type XYZ?
We do not have data for various cell types. Customers report that different amounts of dye may be needed for different types of cell types with various amounts of intracellular antigen. We recommend that you try out various times of incubation and concentrations by live-cell-imaging in your own tissue culture conditions. This optimization must be performed first to get some idea before jumping on to other types of staining such as FLOW, Cell-Sorting, or Tissue staining works.

4. What fixatives to use to get the best result for cells or tissue?
There is no one fixative that we recommend. The dye is designed to be used for a live-cell-imaging purpose. Some researchers used a mild fixative (intracellular antigen not damaged by the fixative). Customers report that the harsher the fixative (formalin) and the age of the tissues can vary the success of the dye binding to the available antigen that is still present in the fixed cells or tissue.

FAQ

FAO Blue (Fatty Acid Oxidation Detection Reagent)

[KOD One] Troubleshooting High-LNA Duplex PCR with KOD Master Mix: A Guided Optimization Approach

LipiORDER [1]

LIVE CELL IMAGING product [1]

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