Can the kit from Invitrogen™ LIVE/DEAD™ Cell Imaging Kit (488/570) be used on fixed cells on a scaffold?
Based on my experience, The Invitrogen™ LIVE/DEAD™ Cell Imaging Kit (488/570) is generally not recommended for use on fixed cells, including those on a scaffold. Here's why:
- Mode of Action: This kit relies on the differential permeability of cell membranes. The SYTOX Green stain readily enters cells with compromised membranes (dead or dying), while the calcein AM stain is taken up by live cells with intact membranes and converted into a fluorescent product. Fixation disrupts cell membranes, potentially allowing both stains to enter all cells, regardless of viability.
- Manufacturer's Instructions: The kit instructions from Thermo Fisher Scientific (Invitrogen's parent company) specify its use for live cell staining. They don't recommend it for fixed cells. This you can find on the outer label.
Alternatives for Viability Assessment on Fixed Cells:
Several alternative methods can be used to assess cell viability on fixed cells on a scaffold:
- Membrane Integrity Assays: Assays using propidium iodide (PI) or 7-aminoactinomycin D (7-AAD) can be used on fixed cells. These dyes penetrate cells with compromised membranes and stain the nuclei, indicating dead or dying cells.
- Metabolic Activity Assays: Some commercially available kits measure metabolic activities of fixed cells. These can be indirect indicators of cell viability.
- Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) assay: This technique identifies apoptotic cells by detecting DNA strand breaks in fixed cells.
The Invitrogen™ LIVE/DEAD™ Cell Imaging Kit (488/570) is specifically designed for use on live cells. This kit employs calcein AM and ethidium homodimer-1 (EthD-1) to distinguish live and dead cells. Calcein AM is a cell-permeant dye that is converted to green-fluorescent calcein in live cells by intracellular esterases, while EthD-1 is a cell-impermeant dye that only enters cells with damaged membranes, binding to nucleic acids and producing a red fluorescence in dead cells.
Since calcein AM requires intracellular esterases for conversion to the fluorescent form, it will not work on fixed cells as fixation typically inactivates cellular enzymes. Similarly, EthD-1 relies on membrane integrity to differentiate between live and dead cells, a condition that is also compromised during fixation.
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