SOLUTIONS FOR STAINING
2% Formaldehyde
Formaldehyde crosslinks proteins, thereby fixing them so that they remain
stable for long periods of time. Laboratory "formalin" contains methanol as
well as formaldehyde and should not be used for flow cytometry because the
methanol increases background fluorescence. Formaldehyde is formed when pformaldehyde crystals are dissolved in solution. Therefore a solution of pformaldehyde will contain pure formaldehyde that is suitable for fixation of
cells for flow cytometry. It is also possible to buy EM-grade pure (methanolfree) formaldehyde in solution and this also is suitable. Cells stained for surface
antigens and then fixed with formaldehyde at 1% (final concentration) should
be stored for at least 24 hours (in the dark and at 4°C) and then read on a flow
cytometer within 2 weeks (although most cells with most stains will probably
last much longer).
Combine 20 gm of p-formaldehyde in 700 ml of phosphate-buffered saline
(PBS) in a 1 liter flask.
Stir in a fume hood until all the solid dissolves. This will take several hours and
can be left overnight.
After the solution is clear, correct the pH to 7.3 and make the volume to 1 liter
with PBS.
Store in a tightly-covered bottle at 4°C.
This solution has indefinite stability; some labs make it fresh weekly and others
use it for months with no problem. To be safe, make it fresh monthly.
Phosphate-Buffered Saline (PBS)
Although PBS can be bought as a mix of crystals or as a concentrated solution,
making it up from scratch is not difficult.
Weight out:
NaCl 8.0g (138 mM)
KCl 0.2g (2.6 mM)
KH2PO4 (anhydrous) 0.2g (1.5 mM)
Na2HPO4 (anhydrous) 0.9g (6.3 mM)
and add crystals to 700 ml of distilled water.
Stir until dissolved.
Adjust pH to 7.3 and make final volume to 1 liter.
Filter through an 0.2 micron sterilizing filter into sterile bottles and store at
4°C.
Staining/Washing (S/W) Buffer
Some labs stain and wash cells in medium (minus phenol red and biotin)
supplemented with 0.1% azide. Alternatively, PBS (or other balanced salt
solution compatible with your cells) supplemented with 1% bovine serum
albumin (BSA) and 0.1% azide is adequate to satisfy most cells throughout the
staining and washing procedure. The BSA goes part way to making the cells
think that they are back home on the farm and the azide is a metabolic poison
that prevents capping and internalization of surface proteins and inhibits
microbial growth. This buffer can also be used for diluting antibodies.
To make S/W Buffer, add
1 gram of sodium azide (NaN3)
10 grams of bovine serum albumin
to 700ml of PBS. Stir gently to avoid foaming of the protein. When dissolved,
check that the pH is 7.3. Make the final volume to 1 liter. Filter through an 0.2
micron sterilizing filter into sterile bottles and store at 4°C.
Blocking Solution
Blocking Solution is important in the staining procedure because it fills the Fcreceptors on cells, thereby preventing irrelevant binding of whole antibodies to
these receptors. Human cells should be blocked with human immunoglobulin.
To make Blocking Solution for human cells,
weigh 120 mg of human g-globulin (Sigma)
and make up to 10 mls with S/W Buffer.
Stir very gently at room temperature until dissolved.
Filter through an 0.2 micron sterilizing filter into vials and store frozen.
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