This is a follow-up to a query I posted several months ago about the 96-well plate automatic loader for the FACSCalibur available from Cytek (Cytek Development, contact Eric Chase 510-657-0102. Website http://www.cytekdev.com/ ). Many people were interested in the unit, but nobody reported any direct experience with it. We have since had the opportunity to use the Automated Microsampler (AMS) for several months (via Ray Lannigan at Tritech Field Engineering, 800-886-7004), and it seems very good overall. Below is a summary of our experience with it. The sampler consists of a robotic plate carrier that fits under the cytometer's sample tube, a control unit that takes about 1 foot of bench width to the right of the cytometer, and a set of wash and waste bottles that fit behind the controller. There are two cables that connect to the cytometer's internal circuitry and a fluidics/pneumatics bundle joining the plate carrier with the control unit. The unit works by positioning the plate below a probe that is connected by a short (3-inch) piece of tubing to the cytometer's sample uptake tube, then lowering the probe into the well and creating a seal around the edge of the well, stirring the contents of the well with a small paddle attached to the bottom of the sample probe, and applying air pressure to the well to drive the sample up the tube. After the sample is taken, the plate carrier moves a washing station under the probe, and the probe is rinsed and backflushed with water from the AMS wash bottle. The user can vary the duration of the rinse and backflush to minimize carryover from sample to sample (minimal in our hands, less than 1 percent with 2-3 seconds for each step). The system requires about 3 sec. of boost time to get the sample to the cytometer and another 3 seconds or so to synchronize the AMS unit with CellQuest (more below). Altogether, it takes about 15-20 seconds per sample in addition to the actual time of sampling and counting, and a 96-well plate takes about 40 minutes of hands-free operation to process if the cell density is reasonable (100,000 cells in a volume of 100 ul). Once we learned how to set it up properly, we were very satisfied with its performance, and we have been able to run through 200+ samples with a minimum of attention. Operating the AMS takes a bit of getting used to. The interface with the computer is somewhat awkward because the software on the AMS doesn't actually communicate with CellQuest. Instead, the user specifies a desired number of events to be saved and a maximum acquisition time, and the AMS takes over control of the mouse and literally moves the cursor over the "acquire" and "save" buttons in CellQuest when these values are met. The AMS counts threshold events that occur after a preset time, and it does not begin counting exactly when CellQuest does, so the number of events saved will vary slightly from file to file and will be close to but not exactly equal to the number of events set on the AMS. With a little practice, it's not hard to keep CellQuest and the AMS synchronized, but it's important to keep CellQuest from terminating the acquisition before the AMS is ready. The main limitation of this setup, aside from the unequal event numbers in each file, is that there is no way to set the number of events based on any parameters that come from CellQuest. In other words, it's impossible to acquire a set number of events that occur within a specific gate. For many applications, this will not be a serious limitation (just acquire more events, up to 60,000 maximum, and gate during analysis in CellQuest), but for analyzing cells representing less than about 20% of the population, this unit will not work well. We have noticed a few quirks. (1) In a few wells, the unit has kept sampling cells beyond the specified cutoff number (e.g., 35,000 when we asked for 5000), and despite consultation with Cytek, we have not been able to solve this problem. This results in strange-looking histograms but does not cause data loss. (2) Occasionally, only 94 or 95 data files are saved for a 96-well plate, or an empty data file was saved. This seems to be related to a failure of the probe to make a good seal around the well (often at the edge of a plate) and may be specific to the unit we've tested (one solution: put standard beads in every row or two to show where the well was dropped). (3) The two units we've tested out have had problems with overheating, which apparently have been fixed by several new design features. We have found Cytek very helpful and responsive to our questions, and willing to redesign parts when problems have arisen. Although this product is quite new, it seems to be a good addition to a lab needing to handle hundreds of samples at a time. The time saved by automatic loading is tremendous. The price, around $15K, is substantially better than the $35K unit that BD will be selling. Laird Bloom Phylos, Inc. 128 Spring Street Lexington, MA 02421 lbloom@phylos.com
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