Can I fix cells after live dead staining?
No. LIVE/DEAD Fixable Dead Cell Stain kits are fixable viability dyes that distinguish live cells from dead cells based on cell membrane integrity and access to available amines. Cells can then be fixed for intracellular antigen detection without loss of original cell staining pattern.
What stain is used for live vs dead cells?
Viability Staining A red and green dye are added to a sample; the green dye penetrates all cells (live and dead), whereas the red dye, which contains propidium iodide, only penetrates cells whose cell membranes are no longer intact (and are therefore dead).
Can you stain dead cells?
TO Iodide, also known as TO-PRO®-1, is a green fluorescent, cell-impermeant nucleic acid stain that can be used to stain dead or fixed cells. TO Iodide, also known as TO-PRO®-1, is a green fluorescent, cell-impermeant nucleic acid stain that can be used to stain dead or fixed cells.
Can propidium iodide be fixed?
Adding propidium iodide or ethidium bromide to unfixed cells can be used to estimate viability of the cells, but use rather less dye and measure quickly. Only dead cells will stain by this method. I agree with Berlinda. If your aim is to measure viability (live cells = unstained), then you cannot fix them.
How do you separate dead and live cells?
One of the simplest methods of cell debris removal is density-gradient centrifugation. Density-gradient centrifugation harnesses a device called a centrifuge that spins a heterogenous mixture at high speeds.
Why do dead cells autofluorescence?
Cells become fluorescent under the excitation by suitable wavelength light. It occurs due to the presence of specific molecules – endogenous fluorophores inside cells, which are originated from mitochondria and lysosomes. This cells’ property is called autofluorescence.
What is fixable stain?
LIVE/DEAD Fixable Dead Cell Stains Protocol The Invitrogen LIVE/DEAD fixable dead cell stains distinguish between live and dead cells in flow cytometry. The dyes covalently bind to intracellular and extracellular amines, allowing the staining pattern to be preserved following formaldehyde fixation.
How do you do a live dead stain?
Protocol
- Thaw vial of dye.
- Dilute LIVE/DEAD fixable dead cell stain by adding 50 µL DMSO to vial.
- Add 1 mL of cells to a flow cytometer tube in protein-free buffer.
- Add 1 µL of diluted stain to cells.
- Mix cells and stain.
- Incubate 30 minutes.
- Wash cells.
- Analyze on flow cytometer.
What does annexin V do?
Annexin A5 (or annexin V) is a cellular protein in the annexin group. In flow cytometry, annexin V is commonly used to detect apoptotic cells by its ability to bind to phosphatidylserine, a marker of apoptosis when it is on the outer leaflet of the plasma membrane.
Are Floating cells dead?
With adherent cells, yes, floating cells are generally all dead. The exception would be shortly after initial plating of the cells, in which case some cells will not have settled to the bottom of the dish.