Cell Fixation and Permeabilization Protocol using 70% Ethanol V.3
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External link: https://www.biolegend.com/protocols/cell-fixation-and-permeabilization-protocol-using-70-ethanol/4252/
Protocol Citation: Sam Li . Cell Fixation and Permeabilization Protocol using 70% Ethanol. protocols.io https://dx.doi.org/10.17504/protocols.io.bacziax6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Created: December 11, 2019
Last Modified: December 11, 2019
Protocol Integer ID: 30841
Keywords: fix perm, ethanol, flow cytometry
Materials
MATERIALS
Cell Staining BufferBioLegendCatalog #420201
Prepare 70% Ethanol and chill to -20°C.Tip: Do not freeze ethanol for long-term storage.
Prepare target cells of interest and wash 2X with PBS, centrifuging at 350xg for 5 minutes
Discard supernatant and loosen the cell pellet by vortexing.
Add 3ml cold 70% ethanol drop by drop to the cell pellet while vortexing.
Continue vortexing for 30 seconds and then incubate at -20°C for 1 hour.
Wash 2X with BioLegend Cell Staining Buffer (Cat. 420201) and resuspend cells at 0.5-1.0 x 107 cells/ml.
Use 100µl cell suspension/staining tube.
Note: Please note that certain markers or fluors may not survive ethanol fixation. Protein-based fluors, like PE and APC, tend to have more difficulty, while synthetic fluors, like Brilliant Violet™, tend to have a higher chance of surviving the process.