Biology Reference
In-Depth Information
3.6 Sample
Collection and
Hematocrit
Measurement
1. Anesthetize the rat (see Subheading
3.3.2
, step 1).
2. Gently lay the rat flat on its stomach upon a sterile support.
3. Using a sterile razor blade make a short transversal cut upon
the caudal vein (see Note 36).
4. Collect the leaking blood using heparinized capillary tubes.
5. Transfer the blood from two capillaries to a test tube contain-
ing 15
3.6.1
Serum Collection
L of 0.5 M EDTA pH 8.0.
6. Gently flip the test tube containing EDTA and the collected
blood once to avoid clotting.
7. Centrifuge the blood sample at 2,000 ´
g
for 5 min in a microfuge
at 4°C.
8. Transfer the serum (the upper phase) to a new test tube (short-
term storage at 4°C; long-term storage at −80°C).
μ
1. Collect one capillary of blood from the rat (see
Subheading
3.6.1
, steps 1-4).
2. Seal one end of the capillary with the sealant and place it on the
holding tray.
3. Centrifuge the capillary for 3 min at 16,000 ´
g
at room tem-
perature using a microfuge equipped with a capillary rotor.
Following centrifugation the blood is separated into two dis-
tinct volumes: the packed cell volume (PCV) and the serum
volume.
4. Measure the length of the PCV fraction and the length of the
total blood volume contained in the capillary using a ruler.
5. Calculate the hematocrit dividing the length of the PCV by the
total length of the blood sample and express the measurement
as percentage.
3.6.2 Hematocrit
Measurement
Table
1
shows the hematocrit values measured in rats treated
with AAV (
46
).
1. Anesthetize the rat (see Subheading
3.3.2
, step 1).
2. Lay the rat on his stomach and place it with the eyes under an
adequate light source.
3. Keep the rat head steady with one hand.
4. Insert a 33 gauge needle (mounted on the Hamilton syringe)
into the anterior chamber of the eye. The anterior chamber is
visible like a clear transparent ring (see Note 37).
5. Aspirate the ACF (about 10 mL) with the syringe.
6. Remove the needle slowly and carefully and transfer the ACF
into a test tube. Store the ACF at 4°C or −80°C for short- and
long-term storage, respectively.
3.6.3 Anterior Chamber
Fluid Collection
Search WWH ::
Custom Search