M. Fero — 4/11/00, updated 4/25/03
Plastic tubes with anticoagulant: PBS + 2 µL anticoagulant for each 100 µL blood to be collected
Anticoagulant: 10% Na2EDTA, pH=7.4.
250 µL of fresh mouse blood in plastic tubes containing EDTA.
RBC lysis buffer (388 mM NH4Cl, 29.7 mM NaHCO3, 25 µM Na2EDTA)
20.75 g. NH4Cl
2.5 g. NaHCO3
0.093 g. Na2EDTA
1 L. H2O
Hema-3 Stain set (Fisher)
EDTA acts as an anticoagulant by chelating Ca++ ions and inhibiting activation of the clotting cascade. If viable cells are needed for culture or sensitive biochemical assays then heparin should be considered in place of EDTA. The lysis buffer is a hypotonic solution that causes osmotic lysis of RBCs, but leaves WBC intact. It must be given at 19-20x the volume of blood to achieve lysis. Extra lysis buffer may be necessary if full RBC lysis is not achieved. An excess of lysis buffer generally will not hurt the WBC. If the WBC need further manipulation after RBC lysis, then wash the cells by centrifugation and resuspension in PBS or media.
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.
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.
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.
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.