Thursday, October 13, 2011

Counting Cells with the EMD-Millipore Scepter 2.0

I recently had the chance to play around with the Scepter 2.0 Automatic Cell Counter from EMD-Millipore.  The Scepter uses the Coulter Volume principle to count cells in a microfluidic chamber connected to a handheld device.   I'm basically using it for things like confirming pre- and post-sort cell counts, as well as counting cells being passaged and primary cells such as PBMCs and splenocytes. The device itself is basically shaped like a pipetteman, and even has a plunger type action which simulates pipetting.

EMD-Millipore Scepter 2.0
To use the device, you need to attach a single-use 40um or 60um sensor, which provides the microfluidic channel through which the sample is passed.  Once attached, you simply hold down the plunger, submerge the sensor tip into a sample volume of ~100ul and then let go of the plunger.  It takes up about 50ul of your sample through an orifice in the sensor and measures the volume of the cells.  It then plots the volume (or diameter) of the cells in a frequency histogram displayed right there on the device's built-in display.  Using a click-wheel on the finger grip side of the scepter, you can adjust the low and high bounds of the histogram in order to remove small (dead/debris) and large (aggregate/larger cells) events.  Once you set these bounds, it displays the event number/mL at the bottom of the display.  The sensor tips are single use and each have a range of cell sizes and sample densities it can handle.  The 40um tip is geared towards cells with a diameter of 3um to 17um and a cell density of 50,000 cells - 1.5x10^6 cells per mL.  The 60um tip can handle cells with a diameter of 6um to 36um and a cell density of 10,000 - 500,000 cells per mL.  The handheld unit can store up to 42 histograms, but this data can be downloaded to a computer and analyzed with the Scepter Software Pro (Mac/Win - Free!).  In the desktop software, you can add info like original volume, dilution factor, sample names etc..  You can also re-gate and overlay histograms to create handy figures.  When you have everything set up, you can export reports in table format which you can open up with Excel or other spreadsheet programs.

Scepter Software Pro Screenshot
So, how does it work?  Well, it certainly counts things very accurately.  Previously, I've found that my MoFlo XDP reports sorted cells really well (at least when the side streams are behaving themselves), and I have lots of data comparing sorter counts to counts using a standard coulter counter or even counting cells on a conventional cytometer using absolute counting beads (which I get from Spherotech, by the way).  The only drawback is you don't get a live/dead report like you might with visual-based systems (e.g. Hemacytometer counts with Trypan Blue, Coulter's Vi-Cel, Invitrogen's Countess, or even Nexcelom's Cellometer).  Sure, you can sort of approximate what's live and dead using volume or diameter as a discriminator, but all the profiles I have been collecting don't really show a clear distinction.  It's certainly not like staining some cells with PI and throwing them on a FACScan with counting beads.  But, with that said, the system worked pretty darn well.  Getting back to the accuracy thing, it matched my counts from a sorted fraction on my MoFlo to within 1%.  That makes me feel good in two ways:  1.  My MoFlo is sorting well, and 2.  The Scepter can actually count really well in a very short amount of time (30 seconds by the way).  I did have one small snafu (described below) which was giving me some really weird results, but outside of that, it performed as advertised.  The one thing that I might have to complain about is the cost of the single-use, disposable sensors.  $3 a piece.  Ouch!

Blue histogram represents a collection in the upright
 position, which leads to low end bubbles showing
 up and screwing up the counts.  The green histogram
 repesents a collection up-side-down and no bubbles
 ending up in the chamber leading to way more accurate
Now, about that snafu.  What I kept finding was after the sample was loaded into the sensor, and then the sample started traveling through the sensor orifice into the counting micro-channel, I kept seeing bubbles creep in there.  The effect of this was I'd start getting these really low volume events piling up near the end of the counting process.  It was a small number of low volume events that I could probably gate out (see figure below), but it still messed things up for me.  Since I was doing a 1:10 dilution (10ul sample, 90ul buffer), when I back calculate (or better yet, let Scepter Software Pro back-calculate for me) the concentrations, I was off as much as 1x10^6 cells (or a 12% swing in total cell counts).  To solve this problem, I made one modification to the collection process.  As soon as the sample was loaded into the sensor (it beeps at this point), I immediately flipped the entire Scepter apparatus upside-down as to force any air that begins to enter the sensor to remain near the tip and not enter the orifice and microfluidic channel.  This got rid of all the air bubbles and my counts became extremely accurate.  In one case, my MoFlo told me there should be 8.02x10^6 cells, and the Scepter counted 8.01x10^6 cells.  This made me happy.  To see this awesome flip move in action, check out the video below.  I apologize for the sound, I was filming this in my sorter room, which has the gentle hum of a twin diesel engine for background noise.  Also, you'll just have to trust me when I say "see the bubbles." UPDATE:  After playing around with volumes a bit more, it's pretty evident that you definitely need 100+ microliters of volume in your tube.  I could get bubbles every time if I only had the requisite 50ul of sample, but if I had 100-120ul, I almost never got bubbles.  With this volume, there's no need to turn the scepter upside-down.

So, in all, I think this product was successful for what my purposes were.  It's small. The counting process is fast.  I can offload the data to my computer, and the counting was very accurate (as long as I remembered to hold it up-side-down to avoid the bubbles).  Will I continue to use it?  I guess it sort of depends on whether or not I can get over not 'knowing' the %live/dead.  For what I'm doing, that's probably fine, but could another option be just as easy and accurate and cheap AND give me live/dead?  To be determined.  I will say that I've used early versions of the Countess and the Nexcelom, and neither impressed me so much as to make me want to buy one immediately.  Hopefully I'll be able to check them out again and perhaps put together a head-to-head review.