6-carboxyfluorescein (6-CF) labels vacuoles

A couple months ago, I tried to use tetramethylrhodamine (TAMRA) as a fluid-phase marker to label vacuoles.  The idea is that the only way this stain could be internalized is if it is pinocytosed, eventually ending up in vacuoles.  If you follow this link, the dye worked OK but not great.  I found a couple other paper that utilized 6-carboxyfluorescein (6-CF) instead of TAMRA to label vacuoles with the same mechanism in mind (pinocytosis).  I tried this stain to see if the labeling was improved over TAMRA.

These are P15 bEnd seeded at 50000 cells/cm2 on SC309 transwells.  I supplemented the media with either 0.6, 0.06 or 0.03 mg/mL 6-CF (from a stock DMSO solution) and allowed the cells to grow in this media for 1 day.  Since 6-CF is green fluorescent, I couldn’t do a Live/Dead stain, so I simply took phase contrast and green fluorescent images.  I found that this dye was extremely difficult to wash off the cell monolayer.  So difficult that I broke the 0.6 and 0.03 mg/mL samples because I rinsed too long and too vigorously.  I was able to rinse most of the dye off the 0.06mg/mL samples (this is also the most common concentration cited in the literature).

The following panels show the phase contrast and 6-CF images by themselves on the top row and then an overlay on the bottom.  These are from 2 different pnc-Si Sepcons.

Sample 1:

Sample 2:

In both samples, there are quite a few bright green puncta that correspond directly with holes in the cells in the phase images.  Although many of the vacuoles did not label green, the % labeled with 6-CF is much higher than with TAMRA.  What I find particularly interesting is that many smaller but also a few of the largest vacuoles all stained green.  I would have expected either/or but not both.  I think <100% labeling efficiency is reasonable because the papers I mentioned typically only showed 1-2 cells with green vacuoles and not an entire field of view.  Therefore, this 6-CF protocol is better for labeling vacuoles than TAMRA and this is further evidence that these are actually vacuoles.  I’m convinced now.