Hi,
I was working on running flow cytometry using Jurkat cells that have uptaken 2 um silica beads.
In order to facilitate delivery of silica beads into Jurkat cells, I coated the silica beads with some cationic liposomes (ones that are often used for RNA transfection).
Although excessive amount of cationic liposomes compromised the cell viability, after reducing the concentration, the cells appeared quite healthy under microscope.
However, when I began to run these cells through a flow cytometer, I found something bizarre happening. Compared to the control samples, the viability dye (amine reactive) signals became very scattered (or broadened) and increased so that it became impossible to gate the live and dead cells. Other signals, although not significantly, also became slightly scattered and increased.
To figure out what might have happened, I tried to run Jurkat cells incubated only with beads and Jurkat cells incubated only with liposomes, but the two samples looked identical to the control.
Thus, I doubt that the aberration was either due to beads interaction with the dyes/antibodies or residual liposomes messing up the staining or readouts.
What I suspect is that, when I look at the cells after incubation with silica microspheres, I see many of them on the membrane of the cells (probably not fully uptaken).
The only two possibilities that I can think of are:
one, the silica beads associated with the cells are causing some wild scattering of the readouts
two, the amine group of cationic lipids remaining on the beads are interacting with the amine reactive viability dyes.
Has anyone done something similar to this? Any advice would be deeply appreciated. I've been doing this experiment for months and have not been able to identify the cause...
Thanks!
- Badges
- Science topic
- Similar topics
- Methodology
- Research Methodology
- Research Papers