Flow Cytometry Laboratory

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-NH4Cl. They 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.

Solutions

Acid Citrate Dextrose (ACD)

Chemical	1 Liter	4 Liters	6 Liters	8 Liters
Dextrose Anhydrous	24.5g	98.0g	147.0g	196.0g
Sodium Citrate Dihydrate	22.0g	88.0g	132.0g	176.0g
Citric Acid Monohydrate	7.3g	29.2g	43.8g	58.4g

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)

Chemical	18 Liters
FTA Hemagglutination Buffer	166.14g

 

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. # 11248)

0.87% TRIS-NH4Cl

Chemical	1 Liter	4 Liters	6 Liters	8 Liters
Ammonium Chloride	8.7g	34.8g	52.2g	69.6g
Tris Base	1.21g	4.84g	7.26g	9.69g

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 cycle.

Protocol

1. Collect blood in ACD, 4 parts blood:1 part ACD. Blood can usually be collected the day before use and refrigerated overnight.
2. 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.
3. 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.
4. 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.
5. 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.