Getting Started
E-mail Mitchell Koptchak to schedule a new sorting appointment or for questions about sorting. For brand new experiments, often users will meet with staff to discuss the purpose and goal of the experiment and offer any advice for preparation or what settings on the machine may work best. For example: What kind of cells are you sorting? How many cells do you need? What concentration should the sample be? How much time is needed? Which fluorochromes?
Cell Prep
Cell samples must be in a single-cell suspension. Clumps cause instability in the flow tip or can clog, causing a delay in continuing your sort and extending the length of your appointment. Stream turbulence will cause lower purities in your sorted tubes. We recommend a concentration around 5 million cells/mL. The sample can be suspended in the buffer that works best with your cell type to keep them alive during the sort. Bring extra buffer so the we can dilute the sample if it's too concentrated (it's always easier to dilute rather than concentrate a sample).
Sort rates
12,000 and 25,000 events per second are the maximum sorting event rates for the 100um and 70um tips respectively. Please keep in mind that event rate is measuring every particle detected by the machine, including any debris not gated out. Your sorted population depends on the percentage of "good" cells in the selected populations. The actual speed of sorting your selected cells will depend on the percentage of positive cells and the concentration of the sample .
Please note that for smaller cell types like red blood cells, we recommend 50psi with the 70um tip. For larger cell types like cell lines we run the machine at 30psi on the 100um tip. This also reduces the risk of clogging.
Clumps
Cell samples must be filtered through 40 um nylon mesh to remove clumps of tissue, etc. Pour your sample through the mesh prior to sorting. We have nylon mesh filter tubes in the lab that we will use at cost ($1/tube) as needed if we notice any clumping in the tube you bring. Rinsing with clear buffer will dilute your cell concentration, but help to avoid loss due to clogging. Filtering often causes loss of cells, but is unavoidable for a trouble-free sort.
Important Point: If filtering through mesh isn’t possible, be sure to call the lab and cancel your sort appointment.
Controls
You should ALWAYS bring an unstained control with your samples, along with single color preps for each different fluorochrome you use in your sorting sample. Bring them in Falcon tubes (see above). We can try to run your samples without them, but we cannot give any guarantee the gates are set correctly without a full set of controls to set voltages and gates.
Collection tubes
Sorted samples can be collected into 1.5mL micro-centrifuge tubes, 12x75mm 5mL FACS tubes, 15mL or 50 ml conical tubes. Bring the tubes of choice with you, containing some collection buffer to cushion the sorted cells (preferably something that your cells will be happy to be sorted into to help ensure as many survive the process as possible). It's best practice to bring a few extra tubes. A clog will result in completely restarting the sort, discarding the contaminated collection tube. If sorting onto a plate (6, 12, 24, 96 well) only one population can be sorted at a time.
Temperature
It is possible to control the temperature of pre and post sort tubes. Please let us know in advance if this is necessary. We can set the temperature anywhere between 4° and 37° C
