Isolation of Cells from Peripheral Blood
Note: the procedures below are optimized for ruminants and pigs. RBC from other
species may not lyse easily with TRIS-NH4
might not even lyse well with water. Alternatives include separation using a
higher density Hypaque-Ficoll (sp. gr. 1.119), which will allow the RBC to pass
through and retain all WBC, or using a BD FACS lysing solution ( Cat. # 349202)
after labeling the cells for flow cytometry.
Acid Citrate Dextrose (ACD)
|Sodium Citrate Dihydrate
|Citric Acid Monohydrate
Dissolve chemicals in Milli-Q water at 80% of the total volume. Adjust pH to
7.0-7.3. Approximately 23g of NaOH pellets will be needed. (Caution: Wear
Gloves). Adjust to the final volume. Sterilize by filtration. Store the
bottles in the refrigerator.
Phosphate Buffered Saline (PBS)
|FTA Hemagglutination Buffer
Add the Buffer to 18 Liters of Milli-Q water, and
adjust pH to 7.2-7.4. Aliquot into 500mL bottles as
needed and sterilize by autoclaving. FTA
Hemagglutination Buffer: (Fisher Scientific Cat. #
Dilute the chemicals in Milli-Q water at 80% of the
total volume. Adjust the pH to 7.2-7.4 and adjust the final volume. Aliquot the
total volume into 500mL bottles and sterilize by autoclaving on the liquid
- Collect blood in ACD, 4 parts blood:1 part ACD. Blood can usually be
collected the day before use and refrigerated overnight.
- Distribute the blood into sterile 50 ml polypropylene V bottom
centrifuge tubes, 35 ml/tube. Fill the tubes with PBS + 20% ACD and mix.
Centrifuge 30 minutes at room temperature at 1500 RPM, no brake.
- If you are doing whole blood lysis (retaining granulocytes), remove
as much of the plasma-PBS-ACD mixture using a Pasteur pipet attached to
a vacuum line. Fill the tube with TRIS-NH4Cl,
mix, and place in 37oC water bath until
lysis of the RBC is complete, indicated by a color change from red to
glassy red-black. Centrifuge 8 minutes at 1000 RPM, full brake, room
temperature. Remove the supernate using the vacuum line as above.
Resuspend the pellet in a small amount of TRIS-NH4Cl,
mix well and fill the tube with PBS-ACD. Clumped dead cells and RBC
membrane debris can be removed by passing the cell suspension through a
filter pipet, made by placing a small amount of nylon wool in the lumen
of a 9" Pasteur pipet and sterilizing by autoclaving. Centrifuge 8
minutes at 1000 RPM, full brake, room temperature. Pour off supernate
and resuspend the pellet in PBS-ACD. Repeat the last washing step until
all platelets are gone.
- If you want to get rid of most of the granulocytes, carefully
harvest the WBC layer (buffy coat) from the tubes at the end of the
first centrifugation and put in a clean 50 ml V bottom centrifuge tube.
Fill the tubes with PBS-ACD, centrifuge 15 minutes at 1500 RPM, low
brake, room temperature. Suction off as much of the supernate layer as
possible without disturbing the WBC layer. Fill the tubes with TRIS-NH4Cl,
put in the 37oC water bath until lysis is
observed, and centrifuge 8 minutes at 1000 RPM, full brake, room
temperature. If necessary, resuspend one more time in TRIS-NH4Cl.
Clumped cells can be removed using a filter pipet as described above.
Wash the cells in PBS-ACD with 8 minute centrifugations at 1000 RPM
until all platelets are gone.
- If you want to get rid of essentially all the granulocytes,
carefully harvest the buffy coats from the first centrifugation and put
into 50 ml glass round bottom centrifuge tubes. Two blood tubes can be
pooled into one glass tube. Bring the volume of each tube up to
approximately 40 ml with PBS-ACD. Using an unplugged 9' Pasteur pipet as
a cannula, underlay each tube with 9 ml Accupaque (sp.gr. 1.086;
Accurate Chemical & Scientific Corp., Cat. # AN-3500). Centrifuge 25
minutes at 1500 RPM, room temperature, no brake. Carefully harvest the
lymphocyte layer from the buffer/Accupaque interface, getting as little
of the RBC pellet as possible. Resuspend the interface in PBS-ACD,
mixing well, and centrifuge 15 minutes at 1500 RPM, low brake. Remove
supernate using vacuum line. The residual RBC can be lysed with TRIS-NH4Cl.