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Fixed or Fresh: What’s better for preclinical flow cytometry evaluations?

Posted on: April 18, 2019

Blood Samples

Flow cytometry analysis is a critical technique for guiding preclinical drug development. Flow cytometry data can provide mechanistic insights and define potential biomarkers that correspond to clinical outcomes. But getting the most out of your preclinical flow cytometry analysis may hinge on how your cell samples are handled, specifically whether you are working with fresh cells or fixed cells.

Fresh Cell Samples:


  1. Cell-1

    Fresh cells may function the most like cells in the body as they are minimally manipulated and their intracellular and cell surface proteins are relatively intact.
  2. Fresh cells could be more viable, thus allowing for the analysis of relatively fragile and rare cell types that do not typically survive processing and fixation.


  1. Fresh cells must be used within a narrow time window, which can make flow cytometry planning and execution challenging, especially if only subsets of samples are available at a given time.
  2. Fresh cells cannot be stored or archived for re-analysis at later times.

Fixed Cell Samples:


  1. Fixation with chemicals such as paraformaldehyde allows for cell samples to be stable for longer periods of time, which provides users with greater control and flexibility in planning their flow cytometry assays.

  2. Fixation typically “freezes” proteins in cells thus making staining of fixed samples more consistent and less prone to variations seen in live cells that are under stress as they are processed and stained.


  1. Fixed cells do not always accurately reflect the function and phenotype of their live cell counterparts
  2. Not all cell types and/or cell markers can survive fixation, thus limiting the scope of cell subsets that can be analyzed.

Consider these pros and cons as you decide how to process your cells for your next flow cytometry experiment.

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