Mon Aug 26, 2019 2:58 pm by mleipold
Hi El-ad,
In my experience:
1. You *do* often save some reagent usage: in many cases, we were able to cut the antibody amount in half. For example, for 10 samples barcoded together, this means we could use 5 tests rather than 10 tests.
However, this did *not* apply in every case. And, the barcode agents (antibody or small molecule) do have a cost themselves: in our hands, using the Fluidigm barcodes, the amount of antibody reagent staining that we saved was basically offset by the cost of the kits. So, the total cost per debarcoded sample basically wound up the same.
2. The main way barcoding saves on machine time is by not having to switch samples. I'd say this saves you 10% on time? Imagine 20min acquisition for a normal sample, then 2min or so involving MilliQ rinse (to minimize carryover) and then getting the next sample going. You can also save a bit of time running Wash solution periodically: if you run Wash at the end of each BC'd sample, you can often time it so that you're there when the target time/event number is hit. Then, you can run Wash while the data processing finishes: this usually takes a couple minutes at least, so you're not losing time running Wash during that.
3. If you can't fix your samples, you can't really run them on CyTOF: even if you're not staining intracellularly (not even Ir), the MilliQ washes will bust your cells. I guess this isn't an issue if you're running with CAS, but we don't.
The main issue with barcoding is that one bad sub-sample will poison the entire combined BC'd sample. This could be due to:
a) Sample clumping. One bad sample clumps the entire BC'd sample, causing you to take a *major* hit on cell recovery (as well as overall data quality, since clumpy samples don't stain or wash efficiently).
b) Poor viability. Dead cells often soak up reagent, which can throw off your effective titer to cause dimmer staining.
c) Debris. This often goes with poor viability, but remember, debris from one sample will cause streaking in the *entire* BC'd sample. This streaking will therefore be raised background in *every* debarcoded sample.
I've had instances of all 3.
d) Somewhat separate: very few people do the "exhaustive" Bodenmiller-like barcoding that maximizes the total number of barcodes. However, if they do, and you have a BC reagent failure, you can *miscode* and confuse your samples.....worst case scenario, miscode a PMA+I as an Unstim.
Barcoding is great, but it is *not* a magic wand.
Mike