Vol. 1; Issue 1

KUMC Center of Biomedical Research Excellence (COBRE) in Novel Approaches for the Control of Microbial Pathogenesis

FLOW CYTOMETRY CORE LABORATORY (FCCL) The mission of the Flow Cytometry Core Laboratory is to provide access to state-of-the-art flow cytometry and related technologies to researchers at the University of Kansas Medical Center and other area institutions. Detailed information regarding our capabilities can be found on-line at: www.kumc.edu/flow. Using the FCCL New users of the FCCL are encouraged to contact FCCL staff to discuss your experiments. Often, we are able to suggest techniques and protocols that will help you answer your scientific questions. Once you are trained on the instruments, we will request access to Meeting Room manager so that you are able to schedule your own instrument time. If there are any difficulties with scheduling or experimentation, always feel free to contact FCCL staff. Contact information can be found at www.kumc.edu/flow/personnel.html.

FCCL News Welcome to the first edition of the FCCL newsletter!! The purpose of this newsletter is to highlight the instrumentation

in the FCCL with the goal of matching the instrument to your scientific questions. In addition, we will address some of the common issues that arise in the course of FCCL-related experiments.

Our exciting news for this issue is that the COBRE grant that provides a substantial level of financial support was funded. This grant will be used to support the FCCL for the next five years as we continue to strive for sustainability. What does this mean for you? Without COBRE, we would be unable to support a user fee structure that is comparable to that of other institutions. To highlight the importance of COBRE and institutional support, we will redesign the invoices so that you will see how COBRE and KUMC support all users on campus. Because COBRE supports everyone, we kindly request that all users add the following statement to all manuscripts containing data obtained from FCCL instrumentation: “We acknowledge the Flow Cytometry Core Laboratory, which is sponsored, in part, by the NIH/MIGMS COBRE grant P30 GM103326.”

During the next five years, we will seek to upgrade and add instrumentation that will support the KUMC scientific community. We always welcome suggestions for new flow cytometry-related instrumentation and upgrades. Further, we will gladly facilitate the preparation of instrumentation grants related to technologies appropriate for the FCCL.

Featured Instrument FACSAria IIu What is it?

The FACSAria IIu, or Aria as we more commonly call it, is a three-laser cell sorter (Fig. 1).

What can it do? The Aria has three lasers, 405, 488, and 633 nm, and can detect light in nine

photomultiplier tubes. Its primary function is to examine complex populations of cells and yield pure populations of cells. The Aria can sort lymphocytes at a rate of approximately 15,000 events per second. For larger cells, the rate is much slower in order to optimize the passage of cells through the instrument. Cells from single populations can be sorted into 96-well plates. Alternatively, the Aria can sort into two 15-ml tubes or four 5-ml tubes. The Aria can purify samples that are simply positive and negative for a single fluorophore or as complex as nine-color samples with intricate gating schemes.

Thanks to a renovated biosafety room within the FCCL facility, we are able to sort human samples. Details regarding the standard operating procedures for this can be found on the FCCL website. Briefly, certification that the samples are unlikely to contain risk groups 3 or 4 pathogens is required.

Fig. 1. The FACSAria IIu.

Vol. 1; Issue 1 How does it work?

The Aria sorts by incorporating cells from the sample tube into a stream of sterile PBS. The stream is interrogated by lasers at the flow cell. The instrument electronics know the whereabouts of each cell as the cell passes through the laser intercepts and determines the specific cells meeting the sort criteria. Fig. 2 shows the image of the droplets generated by the Aria during a sort. A transducer in the instrument vibrates the stream inducing droplet formation. Cells in the stream are incorporated into the droplets. If a cell meeting the sorting criteria is in the last drop before the break off, the instrument will charge that drop and that charged droplet would be deflected into the proper collection tube by the charge plates.

We can adjust the intensity of the vibrations by changing the amplitude and the frequency of droplet formation by adjusting the frequency. The best sorting conditions are high pressure coupled with high frequency. Unfortunately, large cells require low pressures and suboptimal frequency in order to prevent damage to the cells. The Drop 1 and gap measure the location of the drops and ensure that the drops form properly. The instrument calculates a “sweet spot” and variation from this location would be indicative of a failure and cause the instrument to stop sorting.

To purify cells, it is critically important that cells remain in single cell suspensions, as clumps will easily clog the instrument. In addition, if multiple cells are incorporated into a droplet, the instrument will reject both cells, decreasing your yield from the procedure.

A common question that arises in planning a sort is how does the size of the cell affect the quality of the sort. It is important to understand that most flow cytometers are designed to sort lymphocytes, which are small uniform cells. The Aria is capable of obtaining quality results with other cell types, but conditions need to be carefully established. As a general rule of thumb, the size of the nozzle, the opening through which the stream will pass, should be five times larger than the cell size. For cancer stem cells, which can be approximately 15-20 µM in diameter, the 100 µM nozzle is typically sufficient, although some users prefer the 130 µM nozzle. When using large nozzles, it is difficult to obtain a steady stream of droplets. With large nozzles, the pressure under which the stream flows must be very low, in fact the ideal pressure for the 130 µM nozzle is lower than the Aria is capable of reliably sorting. For this reason, it takes longer to establish the stream and the cell yield is typically poorer than sorting with smaller nozzles. Reducing the nozzle to 100 µM allows us to increase the pressure and generate a more reliable stream.

Unfortunately, the only way to ascertain the best sorting conditions for your cells is to work out the conditions. Just as you optimize the number of cells and other conditions for your Western blot, RNA analysis, and other assays, we need to optimize the conditions for your sort. It obviously takes time and resources to optimize your conditions, but the quality of your experiments will benefit. If you sort the same cell type routinely, your future experiments will thank you for a little investment up front.

FAQs about the Aria?

Can the Aria detect stem cell side populations? Without a near-UV laser, we cannot identify cancer stem cell side populations using Hoechst 33342, but we can substitute the dye, DyeCycle Violet (DCV), and use the violet 405 nm laser. The distinct side population is defined in a multi-dot plot analysis of the Red (650 LP) vs. Blue (450/40 BP) emission of DCV, excited by the violet laser (405 nm). We have references for this technique, if you are interested.

What is the best purity expected? Best post-sort purity is obtained when the cells to be sorted are a discrete, bright population. Thus, the answer to this question can vary tremendously.

How is purity and yield balanced? We can bias the instrument to sort for greater recovery or better purity. My cells have low expression of our protein. Can the Aria sort these cells? Marginally fluorescent populations are

difficult to sort. Sorting works best when the population to be sorted is much brighter than its negative control. We advise setting very tight gates on dim cells. Dimly stained cells have fewer attached fluorochromes for the lasers to excite, leading to fewer photons gathered by the light collecting optics. Your yield is likely to be low.

Can the Aria sort cells expressing fluorescent proteins? The Aria is excellent for sorting GFP-transfected cell lines as GFP is maximally excited by the 488 nm laser. The past decade has seen the rapid development of fluorescent proteins. The red protein mCherry is a monomeric fluorescent protein with many advantages; it is

Fig. 2. The droplet profile of a sort. The droplet profile was captured in a screen shot and shows the stream of droplets. We can adjust the amplitude and frequency of droplet formation from this window.

Fig. 3. Pre- and Post-sort analysis of cells labeled with GFP and DsRed. DsRed is visualized within the PE-Texas Red channel. The post-sort purity was >90%.

Pre-sort A B Pre-sort

Vol. 1; Issue 1 optimized for expression in mammalian cells, the protein matures quickly after gene expression (half-life = 15 minutes), and it is photo-stable and resistant to photo-bleaching. Unfortunately, mCherry is best excited by a green to yellow laser line but can be excited by the blue 488 nm laser. DsRed is very similar to mCherry. Fig. 3 shows the pre-sort and post-sort analysis cells co-expressing GFP and DsRed.

Are there any other technologies that could serve a similar purpose?

The other instrument that can be used to purify cell population that is housed within the FCCL is the RoboSep. This fully automated instrument uses magnetic bead-based technology in which antibodies against cell markers are conjugated to magnetic beads. The instrument can then separate cells bound to beads from those that are not. Thus, populations can be separated using positive or negative selection. The RoboSep is particularly well-suited when purifying a single population of cells that can be identified with a single marker.

Featured Technique Annexin V/Propidium iodide staining for dead/dying cells General concept

Plasma membrane phospholipids are not evenly distributed between the inner and outer leaflets. Phosphatidylserine is located on the inner leaflet. During apoptosis, this asymmetry is disturbed and phosphatidylserine migrates to the outer leaflet of the plasma membrane. Annexin V is an anticoagulant protein that binds to phosphatidylserine. Annexin V conjugated to a fluorochrome (commonly FITC, but could be any fluorochrome) will detect apoptotic cells, but some non-apoptotic cells can also bind Annexin V. Propidium iodide can be added to detect cells with permeabilized membranes, a sign of late apoptosis or necrosis.

Hints for getting better results with Annexin V and PI staining for apoptosis

1. Annexin V only binds in the presence of divalent cations (Ca2+ and Mg2+). Removal of the cations results in rapid dissociation of phosphatidylserine and Annexin V. Do not use EDTA in your buffers as it chelates Ca2+.

2. Use caution when detaching adherent cells from culture surfaces as trypsinization can lead to Annexin-binding creating false positives.

3. An overly long incubation period can lead to nonspecific binding of Annexin V.

4. It may be necessary to titrate Annexin V on your specific cell type if the Annexin V signal is too weak or too strong.

5. Cells change shape and shed their plasma membranes as they undergo apoptosis; therefore, gate the cells with caution. Fig. 4A shows the Annexin V FITC and PI staining of apoptotic cells. The apoptotic cells are in purple; they are both Annexin V FITC and PI positive. The necrotic cells are labeled in blue and have lost their Annexin V staining. Fig. 4B shows the Forward (FSC) and Side (SSC) profile for the same cells as in Fig. 4A. The necrotic and apoptotic cells are smaller and slightly more granular as they have lost or are in the process of losing their plasma membranes.

Fig. 4. Using flow cytometry to measure cell death. A) Cells were labeled with Annexin V-FITC and propidium iodide (PI). B) The location of PI- (red), Annexin V+PI+ (purple), and Annexin V-PI+ (blue) cells on a FSC vs SSC scatterplot is shown.

Annexin V vs PI FSC vs SSC A B