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Thank you so much for helping us and spotting the error!

Thank you for your question! As our scaffolds are not see-through, positively stained cells can not easily be visualized using a light microscope as one would usually do. Therefore, the assay can be used qualitatively to visually detect the activity of alkaline phosphatase (ALP) present in the culture and compare this visually during different conditions. If you wish to have a more quantitative measure you can take images of each scaffold and use an image processing program to quantify the amount of staining on the scaffold - potentially by normalizing to an empty or untreated scaffold as well as normalizing to cell number. Alternatively, you can choose an ALP activity assay, where the color change of the staining solution is a measure of ALP activity and then measure the absorbance of the solution after appropriate incubation. We hope this answers your question, otherwise feel free to elaborate!

Thank you for your comment and great question! According to the info supplied with the Invitrogen LIVE/DEAD Cell Imaging Kit (www.thermofisher.com/order/catalog/product/R37601), the cells cannot be used for subsequent assays or repeated measure of viability - for this you would need to have replicate samples. The dye used for detection of the dead cells is a DNA-binding dye, which is toxic for the cells - imaging should therefore be performed immediately after staining and usage of this dye cannot be repeated on the same cell population. Conversely, the dye used for detection of live cells is not toxic, but it will be actively effluxed from the cells within a time range of minutes to hours. Once again, microscopy of the cells should therefore be performed as soon as possible after staining. Nevertheless, as this dye is not toxic to the cells, the live cell staining alone can therefore be performed repeatedly on the same cell population. We hope that answers your question!

@@Ossiform Thank you so much for your detailed answer. That was very helpful.

Great question - thank you for asking! The Live/Dead Cell Imaging Assay can be used both qualitatively to get an idea about the general health of your cell culture as well as quantitatively to e.g., compare toxicity of a treatment. Several methods can be used to obtain a qualitative result: Representative images of random areas can be obtained from the microscope, which can be analyzed in your preferred image processing program. The fraction of live/dead cells can then be quantified manually or using a macro. This fraction can be compared to the number of cells seeded. Alternatively, the cells can be trypsinized (protocol for this here: ossiform.com/CustomerData/Files/Folders/11-pdf/249_cell-recovery-by-trypsination-v1-1.pdf) and dislodged from the scaffold prior to live/dead staining and the fraction of live/dead cells can be quantified using flow cytometry. Note that this particular kit from Invitrogen shown in the video is not suitable for flow cytometry, but other kits are available. Lastly, we have also tested the CellTiter-Glo 3D Cell Viability Assay - you can find the protocol for this here: ossiform.com/CustomerData/Files/Folders/11-pdf/253_cellviability-celltiter-v1.pdf or the RUclips video visualizing this method here: ruclips.net/video/2k2LANW5d2E/видео.html. However, note that this is an indirect measure of cell viability/cell count by measuring the amount of ATP present. We hope this answers your question!

Surgery probably needed but you'd recover faster at least.

@@yksyolcusu8437 I’d have to grow 3.15 inches how would this help/work??? Also how long until this would actually be applied for my unique scenario also what about space between the bones as it’s still growing etc

@@yksyolcusu8437any idea on how it would help? I’d prefer to dodge the surgery