The University of Iowa Carver College of Medicine

Flow Cytometry Facility

Intracellular Staining Methods

Abridged from Jacoberger JW, 1991, Intracellular antigen staining: quantitative immunofluorescence. Methods 2:207-218.

Fixation Method Reagents Use Advantages Problems
Organic Solvent Fixation EtOH
Long time storage

Inactivate cellular processes
No covalent modification of DNA, RNA, protein, or carbohydrate epitopes

Permeabilizes cells
Not good for lipophilic antigens

Cell aggregation
Formaldehyde Fixation Paraformaldehyde Fixation for surface or intracellular staining when followed by lipid solvents Relatively mild fixative

Cell membrane integrity preserved
Serverly broadens DNA histogram peaks when using PI simultaneously with cell surface or intracellular staining
Detergent Lysis Triton X-100
Tween 80
NP 40
Rapid permeabilization

Native conformation of intracellular antigens

Requires fewer washes, preserving limited cell numbers

Requires fewer washes
Cell fragility

Loss of cytoplasmic cell components

High debris component

Limited storage time
Freeze/Thaw Freeze/Thaw buffer Same as detergent lysis Same as detergent lysis Same as detergent lysis
Formaldehyde/Detergent Paraformaldehyde
Triton X-100
Primarily used for simultaneous nuclear antigen and DNA content

Detection of antigens in all cell compartments at higher formaldehyde concentrations
Good DNA histogram CV at low formaldehyde concentrations

Low debris component

Few aggregates
Loss of loosely bound cellular components

Short-term storage
Formaldehyde/Methanol Paraformaldehyde
Retains antigen within the cell better than alcohol alone Cells can be stored indefinitely

Antigens retained better within cell

Low debris component

Few aggregates
DNA histogram CV higher than alcohol alone