7 Things to Consider Before you Sort
By David Stegemeier
Are you planning to run a cell sort on a precious sample? Concerned about recovery? Be sure to review our pre-sort checklist of 7 things experienced cytometrists recommend to improve cell recovery:
- Cell Suspension
- Reduce Cell “Stickiness”
- Monitor Cell Conditions
- Panel Design
- Cell Count
- Cytometer Prep
Note: Always consult your flow core manager before performing your sort, as some steps may not apply and others may need to be adjusted.
1. Properly Suspend Your Cells
Your culture/tissue must be put into a single-cell suspension for flow cytometry. To accomplish this, you may need to use:
- Concentration: Too high of a concentration and your sample will not properly collect; too low and it will take forever to sort. Suggested concentration is typically 1-10 million cells/ml. Consult your flow core manager for their recommendation.
- Pellet & Wash: Spin your cells only as hard/long as necessary. Decant (rather than aspirate) the supernatant, while being careful not to dry the pellet. Then break up and re-suspend into a single cell suspension.
- Suspension Medium: Medium without Ca2+/Mg2+ is essential for maintaining a single-cell suspension. Protein is also important, up to 2% (more causes a clogged sample line).
- Mechanical Disruption: There are a variety of mechanical disruption techniques for making single-cell suspensions. “Racking” is a common way to gently suspend a pellet. Simply rake your tube of cells along a rack. However, the optimal method will likely depend on your specific culture/tissue type.
- Protease Digestion: Enzymatic digestion (e.g. with a protease like trypsin) is a highly effective method to detach adherent cells. The digestion can be neutralized using a protease inhibitor. Since most cells are protease sensitive, it is also recommended to use EDTA to minimize protease exposure.
2. Reduce Cell “Stickiness”
Now that your cells are in a single cell suspension, you need them to stay separated. Accomplish this by using:
- DNase: Damaged cells release DNA into your sample, which can cause cell clumping. It may help to add DNase to your medium to clear any lose DNA.
- Protein Medium: Suspending cells in a protein medium helps keep your sample in a single-cell suspension. Adding 10-50% protein in the catch buffer will also help improve recovery.
- The right collection tubes (and pre-coating):
Cells exit the cytometer in a charged fluid droplet that will interact with charges on the plastic tube wall, resulting in cell death and sample loss. If possible, use polypropylene tubes to help minimize tube wall binding. (Note: some types of irradiated polypropylene tubes have been found to increase cellular autofluorescence.)
Unfortunately, the softer polypropylene tubes may not fit your cytometer. In this case, we recommend using polystyrene tubes that have been pre-coated with a protein buffer solution, such as FBS or BSA. This will help to significantly reduce the charge on the plastic.
3. Keep Your Cells Happy
A happy cell is a healthy cell! Smart researchers understand their sample’s preferred conditions. Always be aware of:
- Temperature: Be aware of how your cells respond to temperature changes.
- pH: Keep pH in mind when formulating buffers and media.
- Humidity: Be sure not to let your cells dry out! Sample tubes should be covered (and protected from light!)
- Nutrients: Protein (FBS, BSA, etc.), glucose, NEAA, or other additives can be essential to cell viability.
4. Filter Your Cells
Avoid clogged nozzles! Before sorting, always pass your cells through an appropriate sized filter to remove any clumps or debris. You may even examine your cells under a microscope to be sure no clumps remain. Remember, manipulation of cells can activate them, which can result in aggregation, so be gentle!
NOTE: Excessive debris in the sample can result in a high abort rate! The final centrifugation step after staining the sample can often be adjusted to minimize the amount of debris in the sample.
5. Panel Design
Proper planning will avoid costly mistakes and headaches down the road. FluoroFinder.com makes panel design easy by helping you to:
- Choose the Correct Reagents: Check that your Fluorochromes/Reagents will work on your cytometer/sample.
- Use Positive and Negative Markers: Including dump channels, negative markers and multiple positive markers will help ensure that the sorted cells are what they are supposed to be.
- Minimize Spillover/Spectral Overlap: FluoroFinder offers the only multi-vendor SpectraViewer to ensure your colors won’t bleed.
Video: Reducing Spectral Overlap
- Use a Viability Dye: No matter how carefully you wash and filter, dead cells will remain in your sample. It is always recommended to include a viability dye in your panel to identify any dead cells.
Video: Using Viability Dyes
6. Count Your Cells
As some cells will inevitably die during the sample preparation process, it is important to know your cell count at the time you run the sample. This also helps to determine the expected recovery of target cells. Be sure to count cells AFTER all staining and processing! Counting cells before or during the staining process, or even right before filtering, can give dramatically inaccurate recovery estimates.
Over-concentrating cells will generally reduce your sort purity, so you may want to dilute your sample if the cells are too concentrated.
7. Cytometer Preparation
Some of these steps may already be performed by your flow core staff. Consult with your core manager for details.
- Check Fluid Levels: Be sure you have a full tank of fluid before starting a sort, and that your waste is empty. If you’re doing a live cell sort, you may need to sterilize fluid at least a day before.
- Sterilization: Everything that is involved in a sterile sort should be sterile. That includes all sample tubes, sorting tubes, racks, etc.
- Machine Warm-Up: Warm-up can take 30-45 minutes and sterilization can add another 45-60 on top of that. (Check with your core manager to see how this could affect your sort time.)
- Adjust Threshold Settings: Increasing your threshold makes it easier to visualize a specific cell population, but effectively blinds the cytometer to any events that fall below the threshold. Setting the FSC threshold above the debris can result in a highly pure sample in terms of the cells sorted, but can result in excessive debris being sorted into the sample tube. Be sure to remain aware of your threshold settings and only use threshold to exclude populations when absolutely necessary.
- Nozzle Tip Size: It is essential that you know the size of ALL the cells in your sample, and to choose a nozzle tip size accordingly. Sorting exosomes can certainly be done using a 50-micron nozzle tip, but not if the exosomes are in a suspension of microglia (>20 microns). The flow community’s general guideline is to choose a nozzle tip size at least four- to five- times the size of the largest cells in the sample.
- Sheath Pressure: The higher the sheath pressure, the faster a sorter can acquire samples, and the sooner it can finish a sort. However, the higher the sheath pressure, the higher the resulting shear forces on the cells, and the greater their velocity when they hit the catch tube. For sensitive samples, lowering the sheath pressure (and increasing the nozzle tip size, when possible) can help maintain cell viability.
NOTE: For optimal recovery, the acquisition speed should be no greater than ~20% of the droplet generation frequency; higher acquisition rates are possible, but result in greater loss of target cells due to coincidence aborts. Many sorters allow for the collection of aborted events into a second tube, whose purity will generally be less than 50%. Research your cytometer’s capabilities or consult your core manager for details.