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Mouse Compete Blood Counts

来源:生物谷  2010/4/23   访问量:2081
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Materials:
250 µL of fresh mouse blood in plastic tubes containing EDTA.
RBC lysis buffer (388 mM NH4Cl, 29.7 mM NaHCO3, 25 µM Na2EDTA)

   
20.75g. NH4CL
2.5g NaHCO3
0.093g Na2EDTA
1L H2O
Hemocytometer
Hema 3 Stain set (Fisher)
 
Hematocrit (Packed red cell volume):
Draw blood in 2 heparinized hematocrit capillary tubes.
Spin 5 min in hematocrit centrifuge. Measure total and packed cell volume.
Calculate packed cell volume as a percent of the total.
 
Red cell count:
Dilute cells 1/1000 in PBS.
Add 10 µL to a hemocytometer. Count the number of RBC per large square.
Calculate: RBC/large square x 1,000 dilution x 10 large squares/µL = RBC/µL blood.
 
White cell count:
Add 10 µL whole blood to 190 µL of lysing reagent (a 1/20 dilution). Mix and incubate 1 min.
Add 10 µL of lysed blood to hemocytometer. Count the number of WBC per large square.
Calculate: WBC/large square x 20 dilution x 10 large squares/µL = WBC / µL blood.
 
White cell differential count:
Spot 20 µL of whole blood near the frosted end of a glass slide.
Smear the drop out across the slide with the end of a second glass slide to obtain a thin film with a smooth feathered edge. Air dry the slide.
Hemastain: 5 dips in fixative, blot dry. 3-5 dips in Solution I, blot dry. 3-5 dips in Solution II, blot dry. Rinse 1 min by placing in a coplin jar under gently running dionized water. Air dry.
Under bright field oil microscopy assess the RBC morphology and perform a differential count on a total of 200 WBC.

Automated cell counts: For automated RBC, WBC and platelet total counts send 0.5 mL of blood (or blood diluted in PBS) in a purple top EDTA tube to the hematology lab. Send specimens before 3 p.m. and call ahead of time. A WBC differential requires more blood.

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