As another installment of the “demoing the plate reader” series, we’re also trying to see how well it quantitates DNA. Probably good that I’m actively involved in this process, as it’s making me learn some details about some of the existing and potential assays regularly used by the lab. Well, today, we’re going to look a bit at DNA binding dyes for quantitating DNA.

We have a qubit in the lab, which is like a super-simplified plate reader, in that it only reads a single sample at a time, it only does two fluorescent colors, and the interface is made very streamlined / foolproof, but in that way loses a bunch of customizability. But anyway, we started there, running a serial dilution of a DNA of known high concentration and running it on the qubit (200uL recommended volume). We then took that same sample and ran it on the plate reader. This is what the data looked like:

So pretty decent linearity of fluorescence between 1ug to 1ng. Both the qubit and plate reader gave very comparable results. Qubit readings missing on the upper range of things for the qubit since it said it was over the threshold upper limit of the assay (and thus would not return a value). This is reasonable, as you can see by the plate reader values that it is above the linear response of the assay.

So what if you instead run the same sample as 2uL on a microvolume plate instead of 200uL in a standard 96-well plate? Well, you get the above result in purple. The data is a bit more variable, which probably makes sense with the 100-fold difference in volume used. Also seems the sensitivity of the assay decreased some, in that the results became unreliable around 10ng instead of the 1ng for the full volume in plate format, although again, I think that makes sense with there being less sample to take up and give out light.

7/3/23 update:

Just tried AccuGreen from Biotium. Worked like a charm. They suggest mixing in ~200uL of DNA dye reagent (in this case, to 1uL of DNA already pipetted in), but I tried 100uL and 50uL as well, and if anything, 100uL arguably even looked the best.

Also, I just used the same plate as Olivia had run the samples on Friday. So in running these new samples, I ended up re-measuring those same samples 3 days later. And, well, it looked quite similar. So the samples for reading are quite stable, even after sitting on the bench for 3 days.

Oh, and lastly, this is what it looked like doing absorbance on 2uL samples on the Take3 plate. Looks perfectly fine, although as expected, sensitivity isn’t nearly as much as Accugreen. That said, you do get more linearity at really high concentrations (4000 ng/uL), so that’s kind of nice.

In moving down the floor and having some more space, we’ll be purchasing a plate reader. Based on previous experience / history, I’ve started by testing the BioTek Synergy H1. We’re going to put it through a number of the more traditional paces (such as DNA and protein quantitation), but we clearly have a bunch of cell-based experiments and potential assays that are worth testing on it. Since I’m curious about some of the possibilities / limitations here, I’ve taken a pretty active role in testing things out.

For these tests, I essentially did a serial dilution of various fluorescent protein expressing cells, to assess what the dynamic range of detection could be. Here, I did half log dilutions of the cells in a 96-well plate, starting with 75,000 cells (in 150uL) for the “highest” sample, and doing 6 serial dilution from there down to 75 cells per well, with a final row with media only to tell us background fluorescence. Everything was plated in triplicate, with analyses done on the average.

For the purposes of not having a ton of graphs, I’m only going to show the background subtracted graphs, where I’ve created a linear model based on the perceived area of dynamic range, and denote black dots showing datapoints that linear model predicts, to see how well it corresponds to the actual data (in color).

The above shows serial dilutions high UnaG expressing landing pad cells. Seems like a decent linear range of cell number -dependent fluorescence there, where we can see perhaps a little more than two orders of magnitude in predictable range. Of course, this is one of the least relevant (but easiest to measure) cases, so not super relevant to most of our experiments.

On the other hand, this is a situation that is far more relevant to most of our engineered cell lines. Here, it’s the same serial dilutions of cells, except we’re looking for histone 2A -fused mCherry. The signal here is going to be lower for three reasons: it’s behind an IRES instead of cap-dependent translation, red fluors are typically less bright than green fluors, and the histone fusion limits the amount of fluorescence to the nuclei, so the per-cell amount of fluorescence is decreased. Here, it was roughly a 20-fold range of the assay down from a confluent well. Maybe useful for some experiments quantitating effects of some pertubation on cell growth / survival (without exogenous indicators!) but obviously can’t expect to reliably quantitate more than that 20-fold effect.

Those are direct cell counting assays (or, well, relative counts based on total fluorescence), but what about enzymatic assays, such as common cell counting / titering assays based on dye conversion. I could certainly see these assays potentially having more sensitivity due to a “multiplier” effect through that enzymatic activity. Well, this is what it looks like for Resazurin / Alamar Blue / Cell Titer Blue.

The above is based on Resazurin fluorescence, where we have a pretty decent 2-log range, although even with these conditions, the confluent wells already saturated dye conversion and lost linearity.

Here, I’m testing a different cell titering dye, CCK-8 / WST-2, which only uses absorbance at A460 as its readout. Here, the linear range of the assay seemed to be a lot less; roughly 1 order of magnitude. I’m not showing resazurin conversion absorbance (A570, A600) here, but it looked pretty similar in overall range as CCK-8.

How about timing for the Resazurin and CCK8 assays? Well, here is what it looks like…. Note, I didn’t do the same linear modeling things, b/c I got lazy, but you can still tell what may be informative by eye:

So clearly, we lose accuracy at the top end but gain sensitivity at the bottom end by having the reaction run over night. This is looking at fluorescence. How about absorbance? That’s below:

Same shift toward increased sensitivity for looking at fewer cells, but same dynamic range window, so we lose accuracy in the more confluent wells as it shifts.

Here, the overnight incubation really didn’t do anything. Same linear range, really.