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Fresh versus Frozen Cells - Considerations for Flow Cytometry

Posted on: March 27, 2019

Dendritic cell and lymphocyte

Flow cytometry assays can only deliver reliable, high quality data if the cells used in the assays are viable and handled properly. Immune cells, including lymphocytes and dendritic cells, can be isolated from whole blood using density gradient centrifugation protocols. These peripheral blood mononuclear cells (PBMCs) can be used as fresh cells for flow cytometry or can be frozen for later use. Consider the pros and cons of using each type of cell preparation when you are planning your next flow cytometry assay.

Fresh Cells

Fresh CellsPros:

  1. Fresh cells often have a greater number of viable cells, which means you will be able to measure more live cell events in your flow cytometry assay. If you are trying to measure a rare cell population, using fresh cells may increase the likelihood you will measure a significant number of cells.

  2. Certain cells are only viable in fresh samples and cannot be reliably detected in frozen samples. Dendritic cells typically do not freeze well so investigators prefer use freshly isolated samples for analysis.


  1. Fresh cells must be used immediately after isolation, which can be a logistically challenging situation depending on the number samples processed and used for flow cytometry staining.

  2. Being dependent on fresh cells can be a challenge for validated assays. Fresh cells must be isolated for each assay so you cannot have a true validation sample for use across locations and at different times.

Frozen Cells

Cryopreservation of cells has it's advantagesPros:

  1. Frozen cells can be cryopreserved at different times and stored until you are ready to run batched samples simultaneously at a later point.

  2. Frozen cells permits greater logistical flexibility, so samples can be frozen and shipped for analysis at another site. Validated samples can also be prepared and stored long term for use in other assays.                                    


  1. Not all cells survive cryopreservation, and different cryopreservation media may adversely affect specific cell populations or markers.

  2. Frozen cells may not behave as expected after thawing - they may produce more cytokines than fresh cells, which would be an artifact of using frozen cells and could lead to scientific misinterpretation.

Your best option is to run pilot experiments before deciding if you need to use fresh or frozen cells. These experiments will save you from making difficult choices with your precious experimental samples.


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