Mouse Compete Blood Counts

M. Fero — 4/11/00, updated 4/25/03

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.