Alkaline Phosphatase Live Stain - FAQs

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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Store AP Live Stain at -20 degrees 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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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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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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AP Live Stain can be used daily throughout the life of a culture without any adverse effects.

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AP Live Stain diffuses from your cells over the course of 2 hours after staining.

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Any fluorescence 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.

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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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After adding AP Live Stain to your culture, incubate the cells for 20-30 minutes at 37 degrees C, and then remove the stain by gently tipping the dish and aspirating the dye.

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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 degrees C and is at the proper pH to ensure cell survival.

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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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Do not add the concentrated 500X stock solution directly to the dish. AP Live Stain must be diluted 1:500 in basal medium, such as DMEM/F-12 (Cat. No. 10565-018). Prior to adding 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. Recommended volumes for preparing the 1X AP Live Stain working solution are given in the manual.

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The diluted dye must be used immediately. Do not store diluted dye for later use.

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Remove the AP Live Stain vial from the -20 degrees 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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There are a few key usage steps around removing the growth medium, diluting the stain, and washing the cells. We recommend this short video (https://www.youtube.com/watch?v=5PGqRhOvwmk&feature=youtu.be) before use.

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

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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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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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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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The iPSC colonies can be easily visualized using alkaline phosphatase stain, such as the Alkaline Phosphatase Live Stain (Cat. No. A14353). In addition, reprogrammed colonies can be selected utilizing live staining with Tra1-60 or Tra1-81 antibodies that recognize undifferentiated iPSCs and enable the identification of reprogrammed cells from a variety of human cell types. Please refer to the user manual for the full protocol (http://tools.thermofisher.com/content/sfs/manuals/AP_Live_Stain_man.pdf).

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ELF 97 substrate is cell impermeant. It may be used on live cells that exhibit phosphatase activity on the surface of the cells, but not intracellular phosphatase activity in live cells. The alternative product for live-cell detection of phosphatase activity is the cell-permeable Alkaline Phosphatase Live Stain (Cat. No. A14353).

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No. The ELF 97 reagent does not covalently attach to any cellular components and may be washed away with any subsequent antibody labeling.

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