To begin, gently invert 10-milliliter whole blood tubes 10 times at room temperature. Using a swinging bucket rotor, centrifuge the tubes at 800 g for 10 minutes at 22 degrees Celsius with the brake on. Then place the samples in a biological safety cabinet and check for the three distinct layers.
Label three five-milliliter polystyrene tubes with letters A, B, and C.Swirl and collect one milliliter of the buffy coat by aspiration and transfer to the tube labeled as A.Then add 60 microliters of 0.1 molar EDTA to tube A containing the buffy coat. Add 50 microliters of the cocktail mix to tube A.Pipette up and down at least three times to mix it and incubate for five minutes at room temperature. After that, add 890 microliters of PBS to tube A and mixed by pipetting at least three times.
Next, vortex the magnetic bead tube for 30 seconds. Add 50 microliters of the magnetic beads to tube A and pipette at least three times to mix the beads thoroughly. Immediately place tube A into the magnet stand and incubate for five minutes at room temperature.
Then carefully pipette the enriched cell suspension into tube B, collecting the clear fraction with no or minimal red blood cells for optimal PBMC recovery. After that, remove tube A from the magnet stand and dispose of it. Next, add 50 microliters of magnetic beads to cell suspension in tube B and pipette up and down at least three times.
Immediately place tube B into the magnet stand and incubate for five minutes at room temperature. Carefully pipette the enriched cell suspension into tube C, collecting only the clear fraction. Remove tube B from the magnet stand and dispose of it.
Then immediately place tube C into the magnet stand and incubate for five minutes at room temperature. Carefully pipette the enriched cell suspension into a labeled centrifuge tube and top up to two milliliters with PBS. Transfer 50 microliters of the cell suspension to a sample cup of the automated cell counter.
Add 450 microliters of PBS for a 1 to 10 dilution cell count.
