How to use Invitrogen LIVE/DEAD Cell Imaging Kit with P3D Scaffolds

NOTE: Error in captions at 01:22. You must add 1mL green staining solution to the 1uL red staining solution.
Video protocol for how to perform Cell viability using Invitrogen LIVE/DEAD Cell Imaging Kit with P3D Scaffolds from @Ossiform Research Line.
Note: This video is made with customized P3D Scaffolds.
Content:
00:13 - Thaw the cell kit at room temperature
00:43 - Aspirate the media from the scaffold
01:09 - Add 200uL media to each scaffold
01:22 - Mix the live/dead cell kit solution by adding 1mL green staining solution to the 1uL red staining solution
02:06 - Add 200uL from the live/dead cell kit solution on the top of each scaffold
02:45 - Cover with tinfoil to prevent it from the light damage and incubate for 15 min. at room temperature
02:59 - Flip each scaffold if the microscope's objective is located at the bottom site of the specimen
03:17 - Evaluate cell viability using flourescent microscope
Make sure to check our latest updated protocols on our website:
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[@antoniovasquez5587]

U guys are doing an amazing job ❤

[@PatriciaAlvaroLlopis]

You are adding 1 mL from the green staining to 1 mL red staining. Captions are wrong.

[@Ossiform]

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

[@iremozbek3593]

Thank you for this video! I have questions though, what is the procedure after we check the cell viability? How many minutes later, should we eliminate the live/dead cell kit solution with media? Is there any toxic effect of this solution on cells, if we keep the solution too long? Should we wash the scaffolds with PBS to remove the solution?

[@Ossiform]

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!

[@iremozbek3593]

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

[@grinsunsharma567]

How to count cells??

[@Ossiform]

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 KZbin video visualizing this method here: kzbin.info/www/bejne/aJyVfXSEjJqXaKc. 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!