MouseCompeteBloodCounts

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)

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.