Jan 25, 2016

Public workspaceFixing Cells with Paraformaldehyde (PFA)

DOI
dx.doi.org/10.17504/protocols.io.c5xy7m
  • Faculty of Medicine Flow Cytometry Facility1,
  • U. of Toronto1
  • 1University of Toronto
  • VERVE Net
  • Sullivan Lab
Verve Team
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DOI: dx.doi.org/10.17504/protocols.io.c5xy7m
External link: http://www.immunology.utoronto.ca/FlowCytometry/protocols/PFA_Fixation.htm?quot;%3EPreparation%20of%20Paraformaldehyde%20Fixative%3C/a%3E%3Cbr%3E%3C/li%3E%3C/ul%3E%3Ch4%3EExternal%20Protocol%20Links:%3C/h4%3E%3Cul%3E%3Cli%3EPurdue%20University:%C3%82%C2%A0%20%3Ca%20title=PurdueProtocols%20href=
Protocol Citation: Faculty of Medicine Flow Cytometry Facility, U. of Toronto 2016. Fixing Cells with Paraformaldehyde (PFA). protocols.io https://dx.doi.org/10.17504/protocols.io.c5xy7m
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
Protocol status: Working
Created: June 22, 2015
Last Modified: March 27, 2018
Protocol Integer ID: 919
Abstract
References:
Becton Dickinson Immunocytometry Systems Source Book (1989) 2.10
Lanier, L.L., and Warner, N.L. (1981) Paraformaldehyde Fixation of Hematopoietic Cells for Quantitative Flow Cytometry (FACS) Analysis. Journal of Immunological Methods 47, 25
C.A. Williams, M.W. Chase, Stabilizes cell membranes and preserves cell morphology. Methods Immunol. Immunochem. New York 5, (1976)
Guidelines
Needed:
Working Dilutions:
Thawed aliquots are stable at 4º C for up to 2 weeks.
Dilute 1 part 2% PFA to 3 parts cells in PBS (eg 60 µl + 180 µl to yield 0.5% final concentration).
Materials
MATERIALS
Reagent16% Formaldehyde Solution (10 ml ampoules)Canemco & MarivacCatalog #0173
Safety warnings
Samples should never be left in PFA overnight. This dramatically increases the amount of autofluorescence your samples. Diluted PFA (2-4% solutions) are only good for 1 week, therefore always date you PFA.
Preparation of Paraformaldehyde from Powder
Preparation of Paraformaldehyde from Powder
Prepare PFA from powder
Protocol
Paraformaldehyde (PFA) from Powder
NAME
Paraformaldehyde (PFA) from Powder
CREATED BY
Verve Team
Allow paraformaldehyde (PFA) powder to come to room temperature
Note
(Stored in refrigerator)
Weigh 10.0 g PFA in fume hood.
Amount10 g
ReagentParaformaldehyde Powder (PFA)Catalog #P6148
Flush container with Argon or Nitrogen to prevent air decomposition of p-formaldehyde.
Dissolve in 475 ml distilled H2O in 60-70º C water bath on hot plate in fume hood.
Amount475 mL
Note
**Do not allow water bath to go over 70ºC (formaldehyde will vaporize!).
While disolving, label 100 X 4 ml and 7 X 12 ml tubes (or other combinations of useful aliquots) with the concentration and date.
After 1 hr add 1 or 2 drops of 5M NaOH.
Duration01:00:00
Note
Cloudy suspension will then turn clear.
Allow to cool at room temperature (@2hours)
Duration01:00:00
Add 25 ml 20X PBS (See Recipie)
Amount25 mL
Adjust pH to 7.3.
Filter, aliquot into tubes and freeze.
Note
Aliquots good for at least 5 years.
Thawed aliquots are stable at 4º C for up to 2 weeks.
Dilute 1 part 2% PFA to 3 parts cells in PBS (eg 60 µl + 180 µl to yield 0.5% final concentration).
Preparation from a Stock Solution
Preparation from a Stock Solution
Dilute with PBS.
Note
Dilute only the amount of PFA you will need per experiment to 4% PFA from the 16% stock
Store the undiluted stock at -20°C until needed.
Note
Open stocks should only be kept for one month
Add and equal volume of the 4% stock to samples to end up with a final concentration of at most 2% PFA.
Note
Fixation can be done from 0.5-2%.
Fix cells on ice for 15-30 minutes on ice,
Duration00:30:00
Note
Fixation can be done from 0.5-2%.
Wash 2x with PBS
Note
Fixation can be done from 0.5-2%.