I want to analyse lysosomal trafficking of a WT bacteria and some KO bacteria in HeLa cells with respect to DQ-BSA red. I want to use a fluorescent cargo as a +ve control for my experiment for quantification purposes. Can you suggest some? Thanks in anticipation.
I'm not sure I completely understand your experimental scheme; do you just want to label the lysosomal compartment of the HeLa?
If so, I've used LysoTracker Green (Life technologies) to stain the lysosomal compartment of HUVEC cells, enabling us to then examine fluorescent nanoparticle fate via confocal microscopy.
No, Actually DQ-BSA-Red itself is used to label Lysosomal compartments. Actually I am looking for any cargo which should transport to lysosome so that I can take as a +ve control if bacteria doesn't co-localize by modulating the trafficking pathways. Hope it make more clear.
I see, that is clearer.
I was thinking you could label the lysosomes with a small molecule dye (like LysoTracker) and then use your DQ-BSA as your endocytosis tracker.
However, in your system where you label lysosomes with the DQ-BSA, you need something that will be endocytosed and not a small molecule, so a nanoparticle or polymer.
One option is a labeled dextran; for example, below is a link to a Life tech product of a dextran labeled with a pH sensitive dye. For receptor mediated endocytosis you could consider a labeled transferrin? HeLa should express the receptor (to my knowledge).
http://www.lifetechnologies.com/order/catalog/product/P10361
Dear Peter,
Thanks for your valuable suggestion. I think labelled dextran will be an good option (thanks for the link) because as far as labelled transferrin is concerned I think it may not reach till lysosomes and recycle back from early endosomes.
Dear pawan you can use GFP-LC3. Its a autophagosomal protein or marker which is present in the autophagosomal membrane. Autophagosomes fuse with lysosome and that you can use in your experiment as a positive control.
You can get that plasmid from addgene.
Fluorescent LDL (e.g. Bodipy-LDL) has been used in the past and works OK. The LDL is endocytosed by the LDL receptor and, overtime, accumulates in a lysosome. You'll probably need to serum starve the cells for a while before treating with the fluorescent LDL and use serum-free media for the uptake. Alternatively there are compounds such as oregan-green dextran which is taken up by fluid-phase endocytosis and then accumulates in a lysosome or you could try transfecting with a GFP-tagged LAMP1- or CD63-expressing plasmid. Both proteins are targeted to lysosomes.