Counterbalance serum and in centrifuge and spin at 3000g for 5 minutes
Prepare ficoll for blood processing: pour ficoll into 50 mL conical tube equal to amount of blood collected (3 tubes ~ 25 mL)
Use serological pipette and slowly pipette the blood on top of the ficoll, allowing it to run down the side of the tube and set on top of the ficoll
Remove serum from centrifuge, counterbalance ficoll/blood conical tube and spin at 400g for 30 minutes
Pipette 0.5 mL of serum into labeled screw-top tubes and 1.5 mL urine into labeled screw-top tubes and set on ice
When ficoll/blood is finished spinning, remove plasma layer using serological pipette and put in 15 mL conical tube, careful not to pick up the buffy coat
Pipette 1.6 mL plasma into Eppendorf tubes and spin in Eppendorf centrifuge at 16000g for 10 minutes
After spinning, pipette 1.5 mL plasma into labeled screw-top tubes, careful not to pick up the pellet, and set on ice
Remove buffy coat with serological pipette from ficoll/blood mixture and add to 50 mL conical tube
Fill 50 mL conical tube to the 50 mL mark with DPBS and spin at 400g for 10 minutes
After spinning, discard DPBS in waste, gently break up pellet with 1 mL DPBS
Again fill 50 mL conical tube to the 50 mL mark with DPBS and spin at 400g for 10 minutes
After spinning, discard DPBS in waste, gently break up pellet with 1 mL DPBS
Using serological pipette, fill 50 mL conical tube to the 10 mL mark and gently pipette up and down to mix
Add 15 μL of DPBS/buffy coat mixture to a 15 mL conical tube
Add 15 μL of trypan blue to the 15 mL conical tube
Again fill 50 mL conical tube to the 50 mL mark with DPBS to the remaining DPBS/buffy coat solution and spin at 400g for 10 minutes
While DPBS/buffy coat solution is spinning, add 15 μL buffy coat/trypan blue solution to hemocytometer and obtain cell count
Count 4 small squares
Multiply this number by 4
Double this number
Move decimal over one spot to the left
Divide number by 5
The resulting number is how many milliliters of freezing media are needed to add to the buffy coat conical tube after the last spin
After the third spin, discard DPBS from DPBS/buffy coat conical tube
Use 1 mL freezing media to gently break up pellet
Using serological pipette, add amount of freezing media needed from the cell count, but subtract one from the pellet-breaking process
Gently pipette PBMC/freezing media solution up and down to mix
Pipette 1.0 mL of PBMC/freezing media solution into labeled Nalgene tubes
Place serum, and plasma tubes in -80, along with PBMCs in the Mr. Frosty