Sample Prep Tips for Improving Flow Cytometry

As with most science techniques, the adage “garbage in, garbage out” is apt when it comes to preparing samples for flow cytometry. Starting with high-quality samples is the first step toward reliable, high-quality data. Here is some expert advice on grappling with challenges that are commonly encountered during sample preparation, along with other tips to boost the quality of your flow cytometry data.

Plan ahead for sample prep

Even though it can be exciting to dive right into a new protocol, don’t be tempted to skimp on careful preparation, even if it means a few more steps or a bit of extra time. Plan all of the prep steps and allow adequate time for them—the benefits of better data will far outweigh the hassle. “Have everything in place so that once you start preparing your samples, you can avoid any delay in processing them and avoid samples sitting out on the bench longer than necessary,” says Sharon Sanderson, Flow Cytometry Application Scientist and Product Manager at Bio-Rad Laboratories. “An excessive time between harvest and staining can lead to poor viability or loss of fragile cells such as granulocytes.” Keep your sample cooled at 4℃ if possible (and appropriate given the nature of your experiment) to reduce necrosis and apoptosis.

Optimizing fixation and permeabilization

Optimizing fixation and permeabilization can be challenging, especially if you are studying intracellular and extracellular proteins simultaneously, as these types of molecules use different protocols. For example, extracellular proteins are accessible to surface staining, but can be adversely affected by permeabilization reagents used to access intracellular proteins.

“Intracellular targets require more complex protocols to fix and permeabilize the cells to allow entry of reagents into the cell,” says Mengsha Gong, Product Manager at Proteintech. “If you are staining for nuclear proteins, you will need an even harsher permeabilization method to penetrate not only the cell membrane but the nuclear membrane as well.” She suggests going easy on fixation if possible, because although it “can be helpful for sample preservation, it can also increase autofluorescence of the cells.”

Optimizing the conditions includes balancing healthy extracellular staining with sufficient access to intracellular proteins. You can research publications to optimize this yourself, or take advantage of the high-quality, commercial fixation and permeabilization buffers available. Gong notes that some antibody manufacturers validate their products with specific commercial buffers. “This makes sample prep and protocol design even easier, because you have a proven method to work with, such as with Proteintech’s FoxP3/Transcription Factor Staining Buffer Kit,” she says.

Making and keeping a single-cell suspension

Creating and maintaining a single-cell suspension is essential for flow cytometers to count cells and markers effectively. Although some samples lend themselves to this easily, others such as solid tissue can be more difficult to render into single cells, so you may need to try several methods to find the best one for your sample type. Researching methods in the literature is a good place to start, but possibilities include mincing the tissue, pushing it through a mesh, digesting it enzymatically, or a combination of these. “You want to preserve the integrity of your cells by harvesting them or dissociating the tissue in a way that does as little damage to cells as possible,” says Sanderson. “If you’re interested in a rare cell type, you may wish to choose gentler methods like neutral protease such as dispase, which is a gentler treatment than trypsin.”

Once you have a single-cell suspension, Sanderson recommends checking it out visually under a microscope—looking for singles instead of clumps, and verifying that you have sufficient cells for your planned experiments (using a cell viability dye and counting with a hemocytometer or automated cell counter is helpful here). “This helps you avoid wasting expensive staining reagents and acquiring data from poor samples,” she says.

Your single-cell suspension might need further encouragement to remain clump-free. “Adding DNAse I or EDTA reduces the formation of clumps of DNA released by dead and dying cells,” says Sanderson. “You can further aid the removal of clumps that could block the cytometer or impair results by passing the preparation through a 70 µm cell strainer prior to sample acquisition.”

Removing contaminating cells

When preparing samples from tissues of mixed cell types, removing unwanted cells is especially important. “An example of this could be ensuring the removal of all muscle from bones when collecting bone marrow and red blood cells, which can make detecting leukocytes by flow cytometry difficult,” says Sanderson. Red blood cells (RBCs) are commonly removed using a density gradient, or using hypotonic lysis, which ruptures RBCs by osmotic pressure. “Water, ammonium chloride, or specialized RBC lysis solutions like Erythrolyse can be used,” says Sanderson. “Care must be taken to ensure sufficient lysis of the RBCs without leaving the cells too long in the lysing solution, as this leads to an increase in cell death of the leukocytes.”

Clinical sampling challenges

Clinical studies running translational assays using fresh blood may encounter challenges during sample transit and storage. The effect of transporting biological samples will depend in part on the sample type, and the assays planned. Precision for Medicine is a global clinical research organization that offers comprehensive clinical trial services to advance drug development, and maintains a global logistics team that recommends options for safe sample shipping. “Challenges tend to arise either when the clinical sites need to do sample handling work or the samples are expected to be in transit for 24 hours or perhaps longer,” says Angelina Bisconte, Senior Director of Translational Biology at Precision for Medicine.

Precision recommends using a blood fixative or stabilizer for samples that need to be assayed in under 8 hours. Fixatives contained within direct draw tubes can provide stability for up to 96 hours. “With available blood stabilizers, the blood samples can be stored in the freezer for months and batch processed,” says Bisconte. “We have had considerable success using commercial stabilizers to enable batched receptor occupancy assays, which are some of the most challenging flow assays.” As with any reagent, it’s important to test that the blood fixative or stabilizer works well in the flow cytometry assay. “We consider it very important to understand what transit of samples does to our assay readouts and how to minimize any issues,” says Bisconte. “Investing the time in working with Precision, so we can scientifically meet the objectives of each clinical study, we can provide the most thorough recommendations to help maximize the quality of the sample that is being received for sample testing.”

Such extra preparation time is never wasted. “Don’t underestimate how important sample preparation is, because if your sample is suboptimal, your results will be suboptimal too,” says Sanderson.