How do you fix cells with PFA for flow cytometry?
– Prepare your cells for flow cytometry (block, stain, wash etc…) – Fix cells on ice for 15-30 minutes on ice, and then wash twice with PBS. – Verify the length of time required to fix the sample type… special considerations may be required for virally infected samples etc.
How do I get rid of autofluorescence in flow cytometry?
The level of autofluorescence can be determined using unstained controls. As there is less autofluorescence at longer light wavelengths, fluorophores which emit above 600 nm will have less autofluorescence interference. The use of a very bright fluorophore will also reduce the impact of autofluorescence.
How does PFA fixation work?
PFA causes covalent cross-links between molecules, effectively gluing them together into an insoluble meshwork that alters the mechanical properties of the cell surface. Previous studies report that the cell surface hardens after fixative treatment [7–10].
Does paraformaldehyde have autofluorescence?
Tissue fixation with aldehyde cross-linkers In terms of fixatives that cause the most autofluorescence: glutaraldehyde is worse than paraformaldehyde, which is worse than formaldehyde. Fixation-induced autofluorescence has a broad emission spectrum, occurring across the blue, green and red spectral range.
How long do you fix cells in PFA?
PFA is commonly diluted to 3.7–5% v/v and is applied to cells for 10–15 minutes.
Do I need to fix cells for flow cytometry?
Do not wash or fix samples prior to flow cytometric analysis. We analyze cells by FACS immediatelly after labeling and we never fix them.
Do you need to fix cells for flow?
It will be safer because your cells are no longer alive; in fact in some labs fixed samples may be mandatory. It is convenient and easy. If the antigen or target you want to look at is intracellular you’ll most likely need to fix your cells in order to permeabilise them allowing the antibody/dye to reach its target.
How do I reduce green autofluorescence?
Historically, the main method that has been employed to lower tissue autofluorescence has been to treat the tissue with solutions of Sudan Black or similar nonfluorescent diazo dyes. These hydrophobic dye molecules will generally bind nonspecifically to tissue sections.
How do I fix autofluorescence?
Use fluorophores that emit in a wavelength further from the autofluorescence compounds in your sample. Typically, far-red wavelength fluorophores such as CoralLite 647 are best for this. Commercially available reagents such as TrueVIEW (VectorLabs), have been shown to reduce autofluorescence from multiple causes.
Does PFA permeabilize cells?
PFA by itself may transiently permeabilize the plasma membrane, but it is unlikely to allow access to the nucleus. So if you want to detect nuclear signals, you should add a detergent to ensure full access of labeling to intracellular compartments, as was recommended above.
Can I use formaldehyde instead of paraformaldehyde?
Paraformaldehyde itself is not a fixing agent, and needs to be broken down into its basic building block formaldehyde. This can be done by heating or basic conditions until it becomes solubilized. Once that occurs, essentially they are exactly the same.