Biology Reference
In-Depth Information
2. l-glutamine is an essential component of cell-culture media, but it is unstable
and decomposes spontaneously in aqueous solutions producing ammonia, a
toxic product of degradation. Buy MEM without glutamine and add l-glutamine
immediately before use. Alternatively there are now some commercially
available, stabilized forms of l-glutamine such as GlutaMAX TM (Invitrogen).
3. When working with mammalian cells, it is crucial to obtain them from a reliable
source and to keep good frozen stocks and records. The American Type Culture
Collection is the best source in North America.
4. Paraformaldehyde or formaldehyde? Formaldehyde (HCHO) is a colorless,
flammable gas with a pungent, suffocating odor. Formalin is a 37% solution
of formaldehyde dissolved in water. Normally, an additive such as methanol
is used to stabilize formalin making it unsuitable for microscopy studies.
Paraformaldehyde, a white powder, is a solid polymer ( n up to 100) of
formaldehyde. Fixative solution is made from paraformaldehyde but must
contain monomeric formaldehyde as its major solute. Dilution with water breaks
up the small polymers but heating and an added source of hydroxide ions is
required to break up the large polymers. As formaldehyde in solution rapidly
forms polymers, it must be made fresh although this can be avoided by storing
a stock (25%) solution at -20 °C and making the working solution immediately
before use. Technically, the fixative we describe here is “2.5% formaldehyde
made from paraformaldehyde”; it is usually incorrectly referred to as 2.5%
paraformaldehyde (PFA) solution (12) .
5. Normal serum is used for blocking non-specific binding sites before adding
primary antibodies in immunofluorescence staining protocols. Ideally, it should
be of the same species as the secondary antibodies, but if good quality highly
cross-absorbed antibodies are used, any normal serum will suffice.
6. Mowiol™ is the trade name of polyvinyl alcohol, a group of water-soluble
polymers manufactured by the alcoholysis of polyvinyl acetate. Mowiol™ comes
in different grades and can be partially or fully hydrolyzed, with partially
hydrolyzed ones being more water soluble. Mowiol™ 4-88 is a partially
hydrolyzed polyvinyl alcohol with low to medium viscosity. Glycerol is added as
a plasticizer and can be added up to 30% (v/v). When thawing Mowiol™, ensure
that it has come to room temperature before using, otherwise small bubbles
will form under the coverslip. Mowiol™ mounting media hardens to form a
stable mount so that, in contrast to liquid mounting media such as glycerol, nail
varnish is not required to seal the coverslips. Alternatively a variety of pre-made
mounting media are available from companies such as invitrogen or sigma.
7. The routine addition of antibiotics such as streptomycin and/or penicillin can
mask low-level contamination and should be avoided. For experiments involving
Salmonella , the cells will in any case have to be cultured in antibiotic-free media
for several hours before the experiment.
8. To achieve consistency and reproducibility, it is essential to adhere to a routine
protocol when passaging and maintaining cell cultures. The following points
should be considered. Even if the original stock is of unknown passage number
 
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