Biology Reference
In-Depth Information
3
Methods
3.1 Preparation
of EPO or CEPO
Concentrated solutions of EPO or CEPO (about 1 mg/ml in PBS)
are stored in small aliquots at −80 °C (10
l each). Each aliquot
can be thawed a maximum of 2 times. EPO or CEPO are adminis-
tered by intraperitoneal injection (i.p.) at the dose of 50
μ
μ
g/kg, in
a final volume of 200
l/mouse. For instance, if the mice weight
20 g, each mouse is injected with 1
μ
μ
g of EPO or CEPO in 200
μ
l
of saline solution (1
μ
l stock solution up to 200
μ
l saline).
3.2
Treatments
EPO or CEPO diluted in saline (see above) are injected i.p. three
times a week. For preventive schedule, they are administered start-
ing 3 days after immunization; for therapeutic schedule, starting at
the onset; for late therapeutic schedule, treatment is initiated 27
days after immunization.
3.3 Preparation
of Emulsion of Antigen
and Adjuvant for
Immunization
1.
Emulsion volume
. Each mouse receives 300
l of emulsion. To
calculate the volume, consider 7-8 mice in excess (2-3 ml in
excess) because there is a waste during the emulsification pro-
cess (e.g., for 20 mice, instead of 6 ml prepare 8.4 ml of emul-
sion, calculated considering 28 mice).
2.
Emulsion composition
. Each mouse receives 200
μ
g of MOG
35-55 in complete Freund's adjuvant (CFA), that is incom-
plete Freund's adjuvant (IFA) supplemented with 8 mg/ml of
Mycobacterium tuberculosis
. The solutions of MOG in PBS
(1.33 mg/ml) and
Mycobacterium tuberculosis
in IFA (8 mg/
ml) are prepared separately, in two different glass beakers, and
then mixed in a 1:1 volume ratio (e.g., for 20 mice, prepare
8.4 ml of emulsion: 4.2 ml of 1.33 mg/ml MOG in PBS and
4.2 ml of 8 mg/ml
Mycobacterium tuberculosis
in IFA).
3.
Preparation of the two solutions.
After calculating the volume
needed as above, prepare the two solutions: (a) in a small glass
beaker dissolve MOG 35-55 in PBS (1.33 mg/ml); (b) in a
second glass beaker prepare a homogeneous suspension of
Mycobacterium tuberculosis
in IFA at 8 mg/ml, using a mag-
netic stirrer.
4.
Preparation of the emulsion.
Emulsify the two solutions in a 1:1
volume ratio with two glass syringes connected with a 2-way
luer lock adapter for 4-5 min, until the solution is firm enough
(see Note 1). Work on ice. Fill the insulin syringes with the
emulsion using the 21-gauge needle (see Note 2). Leave the
syringes on ice for 5 min to settle. Eliminate all the air in the
syringes and refill them again. When the syringes are ready and
contain 900
μ
l of emulsion (enough for three mice) without
air, change the needle again with the 25-gauge one. Keep them
always on ice.
μ
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