Frequently Asked Questions

Ordering Table

Sku Name Size Price Qty
A14353 Alkaline Phosphatase Live Stain 50 µL USD 216.00

About Molecular Probes® Alkaline Phosphatase Live Stain

1. What is Alkaline Phosphatase (AP)?
AP is a phenotypic marker of pluripotent stem cells (PSCs), including undifferentiated embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), embryonic germ cells (EGCs) and Embryonic Carcinoma Cells (ECCs). While AP is expressed in most cell types, its expression is highly elevated in PSCs. Therefore, AP staining has been used to differentially stain PSCs to easily distinguish them from mouse embryonic fibroblasts (MEFs) used as feeders and parental fibroblasts commonly used in reprogramming experiments.

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2. What is Alkaline Phosphatase Live Stain?
The Alkaline Phosphatase Live Stain is a stem cell imaging product that allows users to differentially stain pluripotent stem cells (PSCs). The AP Live Stain utilizes an easy, non-permanent, cell viable protocol for identifying PSCs in your experiments. The stain is provided as a concentrated solution that is diluted in basal medium prior to adding to cells.

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3. How does Alkaline Phosphatase Live Stain work?
The dye is a cell-permeable fluorescent substrate for alkaline phosphatase (AP) that is non-toxic to cells, diffusing out over the course of two hours. Simply dilute the dye in basal medium, apply to cells, gently wash, and the cells are ready for fluorescent imaging.

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4. How does Alkaline Phosphatase Live Stain compare to standard AP assays?
Unlike traditional alkaline phosphatase staining assays, which are terminal, the AP Live Stain allows you to visualize your pluripotent stem cell colonies without destroying your cells.  The alkaline phosphatase substrate in the AP Live Stain is non-toxic to cells and diffuses out in two hours.

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5. How many dishes of cells can I stain with one vial of AP Live Stain?
One vial of AP Live Stain consists of 50 µL of a 500X fluorescein-based dye in DMSO, sufficient for four 24-well plates, twelve 6-cm dishes or four 10-cm dishes. 

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How to use Molecular Probes® Alkaline Phosphatase Live Stain

6. What types of cells can be visualized by the AP Live stain?
This product may be used to stain mouse and human PSCs, as well as embryonic germ cells and embryonic carcinoma cells. 

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7. How can I get the best possible results when using the AP Live Stain?
There are a few key usage steps around removing the growth medium, diluting the stain and washing the cells.  We recommend this short video before use. Alternatively, the subsequent AP Live Stain FAQs address these key points.  

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8. How do I prepare the AP Live stain?
Remove the AP Live Stain vial from the –20°C freezer and thaw at room temperature. Avoid repeated freeze/thaw cycles and aliquot if necessary. Maintain the stock solution and aliquots protected from light in amber tubes and minimize exposure to atmospheric conditions. To prepare a 1X AP Live Stain working solution, dilute the 500X stock solution in DMEM/F-12 (Cat. no. 10565-018). Use the diluted dye immediately. 

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9. Once prepared, how long can the 1X AP Live Stain solution be stored?
The diluted dye must be used immediately. Do not store diluted dye for later use.

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10. How do I add the AP Live Stain to my cells?
Do not add the concentrated 500X stock solution directly to the dish. The AP Live Stain must be diluted 1:500 in basal medium, such as DMEM/F-12 (Cat. no. 10565-018). Prior to adding the AP Live Stain, the growth medium must be removed, and the cells must be gently washed twice with pre-warmed basal medium. The 1X AP Live Stain solution is then added directly onto the adherent cell culture.

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11. How much AP Live Stain do I add to my cells?
Determine the amount of 1X AP Live Stain solution required per culture plate using the table below. Once diluted, add the full amount of solution to the dish.

Table 1 Recommended volumes for preparing the 1X AP Live Stain working solution

Culture Vessel Surface Area (cm2) Volume of AP Live Stain (500X) Volume of DMEM/F-12 (Cat. no. 10565-018)
6-well plate10 cm2/well 3 µL1.5 mL
12-well plate4 cm2/well  2 µL 1 mL
24-well plate2 cm2/well 1 µL0.5 mL
35-mm dish 10 cm2 3 µL1.5 mL
60-mm dish20 cm2 6 µL3 mL
100-mm dish60 cm2 12 µL 6 mL

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12. How many times do I need to wash my cells?
There are two washing steps in the staining procedure. First, the cells are washed twice after the removal of the growth medium. Then, after the cells are stained and the dye is removed, the cells are washed twice to eliminate the excess AP Live Stain and reduce the background signal. Washing should be performed gently with pre-warmed basal medium, such as DMEM/F-12 (Cat. no. 10565-018). Handle the cells aseptically, and carefully add and remove the medium with minimal disruption to the adherent cells during this step to avoid damage to cells.

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13. How do I minimize cell death during wash steps?
It is important that the washes are performed gently, but effectively, to remove excess substrate.  Tipping the dish gently and adding the medium to the corner of the dish rather than directly onto the cells will help maintain viability. This same technique should be used for removal of medium and the subsequent wash steps. It is important to use medium that is sterile and has been pre-equilibrated to 37°C and is at the proper pH to ensure cell survival.

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14. How long do I leave the AP Live Stain on my cells?
After adding AP Live Stain to your culture, incubate the cells for 20–30 minutes at 37°C, and then remove the stain by gently tipping the dish and aspirating the dye.

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15. How do I visualize the stained cells?
After 20-30 minutes, remove the AP Live Stain and wash the cells twice for 5 minutes each with DMEM/F-12 (Cat. no. 10565-018). Following the final wash, add fresh DMEM/F-12 and visualize the stained colonies under fluorescent microscopy using a standard FITC filter. Images should be captured within 10 to 30 minutes following the removal of the dye and the most robust fluorescent colonies should be marked for selection and expansion. Since the fluorescent signal leeches out of the cells and into the surrounding medium as the stain is turned over, we strongly encourage that you visualize and capture images immediately following the final wash for optimal signal detection.

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16. What equipment do I need to visualize AP staining?
Any fluorescent microscope with a standard FITC filter can be used to visualize your stained cells.  Alternatively, the FLoid™ Cell Imaging Station (Cat. no. 4471136) captures high-quality, three-color fluorescent cell images right at your benchtop.  For more information on the FLoid™, visit this page.

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17. How long will it take before the AP diffuses from my cells?
The AP Live Stain diffuses from your cells over the course of 2 hours after staining.

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18. How many times can the same cells be stained with AP Live Stain?
AP Live Stain can be used daily throughout the life of a culture without any adverse effects. 

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19. How long should I wait before using the AP Live stain on my cells again?
Colonies may be restained as early as 24 hours after the initial staining. It is recommended that cells are allowed to recover properly to obtain consistent results.

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20. Can I use AP Live Stain in my reprogramming experiments?
The AP Live Stain is ideal for screening colonies during early stages of reprogramming since it selectively stains PSCs while maintaining cell viability. It can be used in later stages of reprogramming to identify undifferentiated cells for the selection of iPSCs for further cultivation. AP Live Stain was developed specifically for use on live cultures for cell maintenance and is qualified to be free of mycoplasma and bioburden, and exhibits extremely low endotoxin levels.

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21. Can I co-stain PSCs with AP Live Stain and antibodies?
Cells can be co-stained with AP Live Stain and any non-FITC labeled antibody. We recommend, however, that the staining occurs sequentially; stain with surface marker antibodies and appropriate non-FITC fluorophores prior to staining with AP live stain since the antibody staining is semi-permanent and will last for several hours, but AP live stain is transient. Note that although AP Live Stain is rigorously qualified for use in live staining, the surface marker antibodies may not be, and thus PSCs that are co-stained with antibodies may not maintain cell viability and sterility.

For a selection of antibodies and dyes that can be used, refer to this page.

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22. How should the product be stored?
Store AP Live Stain at –20°C in the freezer and thaw at room temperature.  Avoid repeated freeze/thaw cycles and aliquot the AP Live Stain into smaller volumes if necessary. Always protect from light and avoid extended exposure at room temperature and atmospheric conditions.

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Expected results with Molecular Probes® Alkaline Phosphatase Live Stain

23. How should my cells look?
The images below show various cells stained with the AP Live Stain. The differential staining of pluripotent cells easily distinguishes them from the feeder cells.


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24. What should I do if I see staining in fibroblast-like cells?
Since AP Live Stain depends on differential expression of alkaline phosphatase, dim staining of mouse embryonic fibroblasts (MEFs) may be observed. Observe the entire dish to distinguish PSC colonies from individual MEFs that may have high levels of autofluorescence. This will not interfere with your experiment. See image below for an example of this type of staining.

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25. How can I prevent poor or dim staining?
Serum or serum replacement components in the growth medium may cause background and this can result in poor or dim staining. After the removal of the growth medium, gently wash the culture with pre-warmed DMEM/F-12 (Cat. no. 10565-018) for 5 minutes. Aspirate and repeat once before adding the AP Live Stain.  To further decrease background staining, perform 3 separate washes of 5 minutes each with DMEM/F-12 for a total of 15 minutes following the staining, and visualize immediately.

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