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 p-formaldehyde crystals are dissolved in solution. Therefore a solution of p-formaldehyde will contain pure formaldehyde that is suitable for fixation of cells for flow cytometry. It is also possible to buy EM-grade pure (methanol-free) 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 g of p-formaldehyde (Polysciences 00380-250) 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.
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 is important in the staining procedure because it fills the Fc-receptors 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.