I recently ran an experiment where I divided a mouse tumor into several wells of a 96-well plate. Each well was stained with a different panel of antibodies. In only one of the wells, I got the strange pattern you see on the far right, where my events formed a distinct diagonal line with a slope much smaller than expected. If I gate on the events, they are abnormal in the other plots as well. Any help you can give would be great, thank you!!
Hi Nikki,
Did you run the samples in the order of your displayed results (left-good; middle-bad; right-worst)?
Mouse primary samples are very 'dirty/sticky' to the FACS machine. My feeling is the events you recorded for the third sample (right) are accumulated dirt rather than true events. Sometimes, I do get the results you showed when I run a lot of samples. In that case, I will run cleaning solution/FACS buffer until no 'events' appear on plot before I proceed to the next sample. If you gate the events for the third sample on FSC-A vs. SSC-A, they probably appear at the corner of the plot or as a streak. The vital stain will also help to distinguish if they are true living cells.
Are these cells gated on a vital stain such as PI upstream of these plots? Dead cells have much different scatter signal than their live counterparts - maybe these 3 wells died off for some reason?
Also, FSC-A is subject to a scaling factor applied by the instrument to establish a linear range between FSC-A and FSC-H/W. The amount of scaling needed varies between cell types, so a dramatically different FSC-A/FSC-H signature between two samples might suggest that you've isolated a different tumor cell type in those 3 wells?
Hi Nikki,
Did you run the samples in the order of your displayed results (left-good; middle-bad; right-worst)?
Mouse primary samples are very 'dirty/sticky' to the FACS machine. My feeling is the events you recorded for the third sample (right) are accumulated dirt rather than true events. Sometimes, I do get the results you showed when I run a lot of samples. In that case, I will run cleaning solution/FACS buffer until no 'events' appear on plot before I proceed to the next sample. If you gate the events for the third sample on FSC-A vs. SSC-A, they probably appear at the corner of the plot or as a streak. The vital stain will also help to distinguish if they are true living cells.
@Govinda Sharma, each tumor was processed three times (one for T cell markers, one for dendritic, one for myeloid). Each of the tumors that gave the odd pattern in the plot on the right (the T cell panel) gave a normal pattern in the other two panels. So that suggests to me that this is not a characteristic of these individual tumors, but maybe something that went wrong with the instrument?
@Kai Ling Liang, the samples in my figure were not run in order. They were just selected to exemplify the categories into which my samples fell. However, I think you're on to something with the dirty/sticky nature of my samples. My sense was that I had clogged the instrument by collecting so many tumor events in a row. My company purchased this instrument in part because of the 96-well autoplay feature, but unfortunately it seems like I will still need to be live monitoring each sample as it runs so that I can stop it and flush it if something like this should happen again. Thanks!
Nikki
I think you also need to filter your samples prior to acquisition, esp some tumors cells that form clogs, both for your experiment sake and machine's!
Happy experimenting :)
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