Iridium Labeling
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I have some concerns in regards to the Iridium labeling of cells. I've done some testing to see if this labeling is necessary in the surface staining protocol. There doesn't seem to be any difference in the data if I leave it out. CD45 is included in the panel and this seems to be enough for the Cytof to be able to define a cell event. I would like to exclude the Saponin step to avoid any problems this may create in regards to class II tetramer staining. Does anyone have an opinion or experience with not labeling with Iridium?
Re: Iridium Labeling
The CyTOF will register a cell event that in *any* channel:
1) Has a metal signal 3 s.d. above background AND
2) That lasts for more than the minimum cell length (default 10 pushes) but less than the maximum cell length (default 75 pushes)
So, as long as *at least one* of your channels satisfies these requirements per cell event, it will be counted, regardless of whether the Ir intercalator is present.
However:
1) Even in PBMCs, there may be some cells that are CD45-. You may not care about them, but they may be present. If they are not counted, it will potentially throw off your Percent of Parents statistics.
2) The high Ir signal also seems to drive a longer "cell length" for a cell event. In an "Ir-by-Cell_length" plot, the hotspot for an Ir-stained cell is usually centered around 30-40 pushes. If your Ir staining is too dilute, or if you use an marker antibody (sometimes even one as abundant as CD45), the hotspot can start slipping to lower Cell_length numbers. This can decrease your ability to resolve singlets from doublets (well, within the limits of the CyTOF).
In summary: Ir staining is not *required*. But has its advantages.
-Mike
1) Has a metal signal 3 s.d. above background AND
2) That lasts for more than the minimum cell length (default 10 pushes) but less than the maximum cell length (default 75 pushes)
So, as long as *at least one* of your channels satisfies these requirements per cell event, it will be counted, regardless of whether the Ir intercalator is present.
However:
1) Even in PBMCs, there may be some cells that are CD45-. You may not care about them, but they may be present. If they are not counted, it will potentially throw off your Percent of Parents statistics.
2) The high Ir signal also seems to drive a longer "cell length" for a cell event. In an "Ir-by-Cell_length" plot, the hotspot for an Ir-stained cell is usually centered around 30-40 pushes. If your Ir staining is too dilute, or if you use an marker antibody (sometimes even one as abundant as CD45), the hotspot can start slipping to lower Cell_length numbers. This can decrease your ability to resolve singlets from doublets (well, within the limits of the CyTOF).
In summary: Ir staining is not *required*. But has its advantages.
-Mike
Re: Iridium Labeling
for surface straining we actually don't use the saponin solution.
I let the cells overnight 4C in fixator (pfa or cytofix) containing the irridium (tested up to 48h without noticing differences).
DNA signal is good.
I let the cells overnight 4C in fixator (pfa or cytofix) containing the irridium (tested up to 48h without noticing differences).
DNA signal is good.
Re: Iridium Labeling
Regarding Ir staining without perm:
The Ir intercalator does not efficiently enter the cell without some form of perm. The amount of membrane disruption that you get from fixation *can* be enough to give you Ir loading, but it is faster (20min at room temp) and more efficient when the membrane has been perm'd in some fashion (saponin or MeOH being the most common).
If you are perm'ing and want to do an overnight Ir stain, people have found that doing a 0.2x (10,000-fold dilution from the 2000x stock, for example) dilution in PBS seems to work well for getting enough Ir in, without having so much you have uninterrupted "tire track" background in the Ir channels. The DVS website says that the 2000x stock is 500mM; if so, 0.2x = 50uM final concentration.
The Ir intercalator does not efficiently enter the cell without some form of perm. The amount of membrane disruption that you get from fixation *can* be enough to give you Ir loading, but it is faster (20min at room temp) and more efficient when the membrane has been perm'd in some fashion (saponin or MeOH being the most common).
If you are perm'ing and want to do an overnight Ir stain, people have found that doing a 0.2x (10,000-fold dilution from the 2000x stock, for example) dilution in PBS seems to work well for getting enough Ir in, without having so much you have uninterrupted "tire track" background in the Ir channels. The DVS website says that the 2000x stock is 500mM; if so, 0.2x = 50uM final concentration.
Re: Iridium Labeling
Thanks for the responses!
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