Mouse Compete Blood Counts

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. NH4CL2.5g NaHCO30.093g Na2EDTA1L 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.