Stained Fixed Cells Then Fix Again

Gear up Now? Fix Later? - Considerations for the Use of Paraformaldehyde Fixation in Flow Cytometry

Sometimes in the middle of a flow cytometry experiment, you have to set up your samples. In that location's a variety of reasons you'll need to ready samples including, but non limited to:

• Staining intracellular targets (e.one thousand. − intracellular cytokine staining, phosphorylation targets) - the cells demand to be stock-still prior to the permeabilization of the cells.
  
• Convenience − for scheduling purposes, you need to fix the samples in the centre of an experiment so you tin can go along to clarify them later.
  
• Disinfection − when you lot're working with infectious samples (i.east. HIV-infected patient samples), it's a routine procedure to pre-fix the sample prior to analysis for sanitation purposes.
  
• Timepoint − when y'all're looking at a progressive biological process over a time-form (e.thou.- phosphorylation of Protein A, X hours after stimulation Y), you'll want to "freeze" all processes using fixation so you get a more than authentic snapshot of what'southward going on at different timepoints.
  
• It's Saturday night - your buddy Chad is having a birthday fustigate at the Legendary bar downtown starting at 8pm. It'south eight:30 already, you're in the lab, and take just finished harvesting your samples. I mean… yous have no choice, right?

Despite all of these unlike reasons for you lot needing to fix your samples, have you considered factors, such every bit:

• • What concentration of fixative should I use for my experiment?
    
  • When in your staining procedure should you lot set your sample in the duration of your protocol?
    
  • Ultimately, how can these decisions potentially affect my catamenia staining?

In this blog, we'll explore these questions related to fixation, and perhaps some nutrient for thought for your next period cytometry experiment when yous run into a fixation dilemma.

For the purposes of this blog entry (more to come on the different types of fixatives...), nosotros'll mainly focus on the use of paraformaldehyde/formaldehyde, usually referred to as PFA, which is the virtually commonly used fixative in flow cytometry. PFA specifically refers to the solid, polymer form of formaldehyde (and dissolves into monomeric formaldehyde in aqueous solution), and so what's usually referred to canonically as "PFA fixation" is actually using the monomeric, formaldehyde solution. In this instance, PFA and formaldehyde are synonymous and interchangeable.

A solution ranging from 1-4% PFA is typically used for fixation of samples for flow cytometry. In the instance of sanitizing infectious samples, concentrations equally low as 0.37% can effectively disinfect samples from HIV-infected patients ^ane . Incubation for up to 45-60 minutes with one% PFA, and 15-20 minutes with four% PFA (e.grand. BioLegend'due south buffer) is sufficient to fully prepare the cells, and the cells tin either exist used for downstream processing (permeabilization for intracellular targets) or stored for time to come analysis at this phase.

During a routine flow cytometry staining procedure that involves surface marking staining, there are several steps where y'all tin make up one's mind to set your samples - prepare after surface marker staining, or fix before .

In some instances, the decision for when you lot tin set is... stock-still. For instance, if you're analyzing phospho-targets, bounden of certain surface antigens past antibodies can alter intracellular signaling pathways shortly subsequently contact, thus leading to an artifact in your phosphorylation results. In which instance, you lot should fix the cells before the improver of the surface antibodies (which is why our recommended protocol to use for our True-Phos™ Perm Buffer to analyze intracellular phosphorylation targets is designed so that the fixation step precedes other procedures). Each has its own benefits and drawbacks, but here's a couple of things to sentry out for in each example.

Fixation BEFORE staining - epitope alteration:

Due to the nature of fixatives, they can cause antigen epitope structures to be altered, which might return the antibodies unable to demark to their targets. Below are some examples of antibodies demonstrating loss of signal when stained on stock-still cells:
Representative plots for target cells stained with (Postal service-Fixation) or without 4% PFA fixation.

Since this effect depends on the clone of antibody y'all're using, you will want to look effectually to meet whether there's any information pertaining to the antibody you're using. However, yous might be in luck! We've compiled a set of information and information (where available) on whether a particular antibody's binding is affected by 4% PFA fixation, which can be found on our Fixation webpage. For clones not listed on our site, yous may want to consult the literature.

Fixation Subsequently staining - fluorophore stability

Upon encountering fixative, fluorophores that are conjugated to the antibodies can potentially lose some of their signal. Poly peptide-based dyes like PE and APC, and especially their tandems (i.east. PE/Cy7 and APC/Cy7), are more than susceptible to the quenching of signal upon encountering fixative solutions, though in most cases they should be able to withstand a standard, 1-4% PFA fixation. Even so, harsher fixatives such as alcohols may be more detrimental to such fluors. In which case, we'd recommend leaning towards the use of synthetic dyes, such as Alexa Fluor® and Brilliant Violet™ (more specifically, BV421™ and BV510™) for your experiments, which will be able to withstand these types of fixation conditions more than robustly, or consider staining after fixation.

To combat the issue of tandem breakup upon fixation, BioLegend has developed the FluoroFix™ Buffer - a PFA-based fixative buffer specially formulated to mitigate signal quenching by sensitive fluorophores.

And then now that you've heard all almost different considerations when it comes to fixing your samples for your flow cytometry experiments, information technology'south time to go fixin'!

Do you lot accept any other questions regarding fixation procedures for your flow cytometry work? Any additional tips and considerations to add hither? Let us know at tech@biolegend.com!

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Source: https://www.biolegend.com/en-us/blog/fix-now-fix-later-considerations-for-the-use-of-paraformaldehyde-fixation-in-flow-cytometry

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