Flow Cytometry Sample Preparation Basics

Author: Mike Blundell | Reviewer: Chloe Fenton

There are many considerations for sample preparation to optimize your flow cytometry. The first one is to take into account your starting sample. Frozen cells will need to be treated differently to an adherent cell line for example; however there are some basic rules that can be followed for most samples that will help you optimize your experiments.

What Is Flow Cytometry Sample Preparation?

Flow cytometry sample preparation refers to the processes used to convert biological samples into a single-cell suspension suitable for flow cytometric analysis.

The quality of sample preparation has a direct impact on cell viability, data quality, reproducibility, and the ability to accurately identify populations of interest.

Although preparation methods vary according to the sample type and experimental objectives, the fundamental principles discussed below apply to most flow cytometry workflows.

Key Principles of Flow Cytometry Sample Preparation

1. Defrost cells as quickly as possible and remove the DMSO by resuspending in a large volume of cold media or PBS containing BSA or FCS. Cells may need to go into culture after defrosting to restore epitope expression.
2. Adherent cells may need gentler treatments than those required for passaging. Trypsin is a harsh treatment that can often destroy cells, creating lots of cell debris, as well as destroying epitopes. More gentle methods of creating single-cell suspensions may be needed.
3. Take care not to be over aggressive when using mechanical disaggregation of tissues such as spleen or lymph nodes. Filtering the sample can help to remove any unwanted clumps of cells.
4. The extraction of some cells from secondary lymphoid tissue, e.g., F4/80-positive macrophages and follicular dendritic cells (DCs), requires additional enzymes such as collagenase or liberase. However, there may also be some unwanted removal of epitopes so optimization may be required.
5. Remove any unwanted contaminating material. For example, when flushing bone marrow from bones, remove as much muscle as possible. Again, filtering can remove any unwanted bone and muscle.
6. Use the appropriate anticoagulant for peripheral blood. EDTA should not be used when detecting intracellular cytokines or some surface markers that require Ca²⁺ ions, such as integrins.
7. Removal of contaminating red blood cells from peripheral blood samples can be performed using hypotonic lysis using a red cell lysis buffer such as Erythrolyse Red Cel Lysing Buffe (BUF04B). Care needs to be taken not to leave the samples for too long in the buffer. Alternatively, a density gradient can be used. After centrifugation, leukocytes are trapped at the interface of the higher density liquid whereas red cells pass through. Unfortunately, granulocytes also pass through the interface so this method is not suitable if this is the cell population you are interested in.
8. Avoid vortexing and excessive centrifugation of your samples and take care to avoid leaving the cells as a dry pellet. Excessive bubbles when resuspending can increase cell death as can suspension at high concentrations. Keeping your cells on ice can improve the viability as this slows down necrosis and the cell metabolism.
9. All sample preparation should be as short as possible, as the time taken to prepare your cells can have a large effect on the cell viability.

Common Sample Types Used in Flow Cytometry

Flow cytometry can be performed on a wide variety of biological samples, including cultured cells, blood, bone marrow, lymphoid tissues, tumours, and dissociated organs.

Each sample type requires a slightly different preparation approach; however, the key principles outlined above remain applicable regardless of the starting material.

Learn More About Flow Cytometry Sample Preparation

If you would like to dig deeper into this subject, take a look at our flow cytometry sample preparation page, which includes a range of extremely useful protocols.