When using the LIVE/DEAD BacLight kit for bacterial viability, with SYTO 9 and propidium iodide (PI), why are some cells detected with both dyes? Shouldn't live cells be green and dead cells be red?
SYTO 9 dye will enter all cells, live or dead. PI only enters cells with compromised membranes (dead cells or damaged cells). First, perform single color staining and examine under both filter sets. Longpass filters or the use of too much dye may result in excessive bleedthrough of the green dye emission into the red channel and the emission of PI in the green channel. Use narrower bandpass filters if possible, and use lower concentrations of the dye. Some live cells may take up PI by engulfment; avoid extended incubation times with the dye. Apply PI to a live cell culture and optimize incubation times to limit engulfment.
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I want to use propidium iodide and SYTO 9 to do LIVE/DEAD testing of a bacterial sample. Will anaerobic conditions adversely affect the assay?
Oxygen content should not affect the binding of propidium iodide and SYTO 9 to nucleic acids. SYTO 9 will label all cells, and propidium iodide will label only dead cells or cells with a compromised membrane.
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I have a LIVE/DEAD BacLight Bacterial Viability kit that has SYTO 9 and propidium iodide in it. Will I be able to stain eukaryotic cells that have engulfed bacteria and determine if the bacteria are alive or dead using this kit?
Unfortunately, no. SYTO 9 will label the nuclei of live or dead cells, including the eukaryotic cells. Propidium iodide is cell impermeant, and will only enter dead cells. If the eukaryotic cells are dead, they will label with propidium iodide as well. If the eukaryotic cells are alive, propidium iodide will not be able to enter and thus will not label the bacteria inside, whether the bacteria are alive or dead. We are not aware of any way to do a viability assay of bacteria once they have been engulfed by cells.
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When using the Live/Dead BacLight Bacterial Viability and Counting Kit, for flow cytometry, some cells seem to have both red and green signal. Are these cells dead or alive or dying?
The green dye in the kit will label all the cells as it is a cell-permeant nucleic acid stain. The red dye is not cell permeant, and will only stain the cells with compromised membranes (dead cells). Therefore, any cells with red signal will be considered dead. It is possible that you will have some cells that are only red, some that are red and green, and some that are only green. Sometimes the red dye will displace the green dye. In any case, any red cells are dead.
Also, the green dye emission may bleed through into the red channel. Do a single-color staining and examine under both green and red filter sets to determine the level of bleedthrough. To avoid this bleedthrough, use a lower concentration of dye, and, if possible, use narrow bandpass filters.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
How can I count my bacteria by flow cytometry?
There are several options. We have two fluorescence based kits that are useful for bacterial counting: Live/Dead BacLight Bacterial Viability and Counting Kit, for flow cytometry (Cat. No. L34856) and Bacteria Counting kit, for flow cytometry (Cat. No. B7277). Another option is the Flow Cytometry Sub-micron Particle Size Reference Kit (Cat. No. F13839).
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
