5. 35 μm nylon mesh.
6. Compensation beads for antibodies [e.g., CompBeads from
BD Biosciences].
7. Compensation beads for viability dye [e.g., ArC Amine Reactive Compensation Bead Kit from Thermo Fisher Scientific].
8. Cell sorter with suitable number of channels, using a 70 μm
nozzle [e.g., FACSAria III from BD Biosciences].
3 Methods
3.1 PBMC Isolation
and Cryopreservation
Due to the nature of human studies, the collection of blood samples is subject to the availability of the individuals to be sampled and
trained phlebotomy personnel. Blood sampling may also take place
a considerable distance from the researchers. Therefore, blood
samples are often processed by isolation and cryopreservation of
peripheral blood mononuclear cells (PBMCs), which can then be
thawed for downstream analysis. This protocol was designed using
cryopreserved PBMC samples ranging in storage time from several
months to over 10 years but could easily be adapted for use on
freshly isolated PBMCs.
1. Transfer blood samples from collection tubes to 50 mL centrifuge tubes and dilute with an equal amount of PBS (see Note
1).
2. Pipette 15 mL of room temperature Histopaque into a 50 mL
centrifuge tube. Carefully layer no more than 30 mL of the
diluted blood on to the Histopaque layer. Repeat for as many
tubes as needed for the complete diluted blood sample.
3. Centrifuge the layered blood for 20 min at 800 Â g, ensuring
that the brake is disabled.
4. Carefully remove the tubes from the centrifuge. Using a serological pipette, remove as much of the top layer of plasma as
possible without disturbing the thin band of PBMCs below.
The plasma can be discarded or stored for further analysis.
5. Using a Pasteur pipette, transfer the thin PBMC layer below
the plasma to a new 50 mL centrifuge tube.
6. Top up the tube to 45 mL with 1% PBS/BSA and centrifuge
for 10 min at 200 Â g at room temperature, with the brake
turned on.
7. Remove the tube from the centrifuge and pour off the supernatant. Resuspend the cell pellet in 45 mL of 1% PBS/BSA and
centrifuge for 10 min at 200 Â g at room temperature, with the
brake turned on.
22
Christopher L. Pinder et al.
6. Compensation beads for antibodies [e.g., CompBeads from
BD Biosciences].
7. Compensation beads for viability dye [e.g., ArC Amine Reactive Compensation Bead Kit from Thermo Fisher Scientific].
8. Cell sorter with suitable number of channels, using a 70 μm
nozzle [e.g., FACSAria III from BD Biosciences].
3 Methods
3.1 PBMC Isolation
and Cryopreservation
Due to the nature of human studies, the collection of blood samples is subject to the availability of the individuals to be sampled and
trained phlebotomy personnel. Blood sampling may also take place
a considerable distance from the researchers. Therefore, blood
samples are often processed by isolation and cryopreservation of
peripheral blood mononuclear cells (PBMCs), which can then be
thawed for downstream analysis. This protocol was designed using
cryopreserved PBMC samples ranging in storage time from several
months to over 10 years but could easily be adapted for use on
freshly isolated PBMCs.
1. Transfer blood samples from collection tubes to 50 mL centrifuge tubes and dilute with an equal amount of PBS (see Note
1).
2. Pipette 15 mL of room temperature Histopaque into a 50 mL
centrifuge tube. Carefully layer no more than 30 mL of the
diluted blood on to the Histopaque layer. Repeat for as many
tubes as needed for the complete diluted blood sample.
3. Centrifuge the layered blood for 20 min at 800 Â g, ensuring
that the brake is disabled.
4. Carefully remove the tubes from the centrifuge. Using a serological pipette, remove as much of the top layer of plasma as
possible without disturbing the thin band of PBMCs below.
The plasma can be discarded or stored for further analysis.
5. Using a Pasteur pipette, transfer the thin PBMC layer below
the plasma to a new 50 mL centrifuge tube.
6. Top up the tube to 45 mL with 1% PBS/BSA and centrifuge
for 10 min at 200 Â g at room temperature, with the brake
turned on.
7. Remove the tube from the centrifuge and pour off the supernatant. Resuspend the cell pellet in 45 mL of 1% PBS/BSA and
centrifuge for 10 min at 200 Â g at room temperature, with the
brake turned on.
22
Christopher L. Pinder et al.