Peripheral blood is the primary source of lymphoid cells for investigations of the human immune system. Its use is facilitated by centrifugation with density gradient media such as Lymphoprep™ or Ficoll-Hypaque™ - a simple and rapid method of purifying peripheral blood mononuclear cells (PBMC) that takes advantage of the density differences between mononuclear cells and other elements found in the blood sample.
Mononuclear cells and platelets collect on top of the layer of density gradient medium because they have a lower density; in contrast, red blood cells (RBC) and granulocytes have a higher density than the density gradient medium and collect at the bottom (Figure 2-1). Platelets are separated from the mononuclear cells by subsequent washing or by centrifugation through a foetal bovine serum (FBS) cushion gradient that allows penetration of mononuclear cells but not platelets.
Equipment
Table 2.1 Table of equipment for PBMC separation
Refrigerated centrifuge with swinging bucket rotor -80˚C freezer
Certified class II microbiological safety cabinet (‘hood’)
Cell counting equipment – Trypan Blue, disposable plastic counting chamber, microscope
Automated cell counter e.g. Muse Cell Analyser Set of micropipettes
Sterile individually wrapped 5 mL, 10 mL and 50 mL serological pipettes Sterile plastic Pasteur pipettes (typically 3mL / 4mL)
Sterile polypropylene conical centrifuge tubes, 15 mL and 50 mL volumes Electric Pipette-Aid
Sterile 2.0 mL polypropylene cryovials with screw cap and O-ring
Freezing container, for example ‘Mr Frosty’ by Nalgene®, catalogue number C1562-1EA
Table 2.2 Table of reagents for PBMC separation
Reagenta Supplier Catalogue No.
Sterile D-PBS Gibco 14190-136
RPMI 1640 Gibco 11875-093
FBS (or FCS)b Sigma F9665
200mM L-Glutamine Gibco 25030-024
100X Penicillin/Streptomycin solution Gibco 15140-122
Lymphoprep Axis Shield NYC-1114547
DMSO Sigma D2650
DNase I Sigma DN25
a Listed reagent or equivalent was used
Reagent Preparation
All reagents were prepared in a Class II hood using aseptic technique. See Appendix 2 for details on reagent preparation.
Density Gradient Separation
Fresh heparinised blood was placed into appropriately labelled 50 ml sterile conical centrifuge tubes. 50 mL Falcon tubes were labelled with the subject number. The appropriate volume of Lymphoprep was aliquoted into each Falcon tube, allowing 20-25 mL Lymphoprep per 20 mL blood. 20 mL blood was slowly layered over the Lymphoprep solution into each tube. To maintain the Lymphoprep/blood interface, the centrifuge tube was held at a 45° angle and the blood pipetted along the side of the tube.
The Falcon tubes were centrifuged for 20 min at 900 × g, at 20°C ± 2°C, with no brake. Using a sterile pipette, the upper layer that contains the plasma and most of the platelets was carefully and slowly removed and discarded. Using another sterile pipette, the mononuclear cell layer was transferred to another 50 mL centrifuge tube. Pasteur pipettes were used for this step. The cells were washed by adding excess sterile R0 media (∼3 times the volume of the mononuclear cell layer) and centrifuging for 10 min at 900 × g, 18° to 20°C.
The supernatant was removed, and the cells re-suspended in sterile R0 by gently tapping the tube with an index finger, and the wash was repeated once, at 200 x
g, to remove most of the platelets.
The mononuclear cells were initially re-suspended in 10 mL R10, pipetting gently up and down to get a single cell suspension. Once re-suspended, the cells were topped up with R10 to a volume equivalent to the original volume of blood.
The cell concentration was determined using both an automated cell counter (Muse Cell Analyser) and manual haemocytometer method. When using the manual method, 10 µL cell suspension was mixed with 10 µL Trypan Blue and
the mixture was aliquoted into counting chamber. The optimal cell concentration for counting generated 50-100 cells per counting field.
The number of PBMC per mL blood was determined by using the following method: number of live cells in field x 104 (x 2 if have used trypan blue at 1:1 ratio). The viability was determined by Trypan Blue exclusion using the manual haemocytometer method (Viability = (number of live cells / total number of cells) * 100).
Blood obtained from a healthy donor typically yields 1-2 x 106 mononuclear cells per ml blood. 60-70% are lymphocytes, with viability > 95%. Platelet count is < 0.5% total platelet content of original blood sample. If there was excess erythrocyte contamination, pure mononuclear cell populations were obtained either by subjecting the cells to a second cycle of gradient separation as described above, or by lysing the erythrocytes with red cell lysis solution (such as ‘ACK Lysing Buffer’ by Gibco, Cat. No. A1049201). A cell count was repeated following red cell lysis.
Freezing Procedure
Cells were frozen in 1 mL aliquots of 10-12 x 106 cells/mL. The volume of Freezing Mix required to freeze the cells at the appropriate concentration and the number of cryovials needed was determined. Labels included the subject ID code, study number, visit number, sample date and the concentration of cells. The required number of cryovials were pre-chilled.
After counting the cell suspension, the cells were centrifuged at 200 x g for 7 minutes at 2-8°C. The Freezing Mix was prepared as follows: 1 mL DMSO to 4 mL RPMI. The Freezing Mix was made daily as required and stored at 2-8°C or on ice until time of use. The supernatant was removed, and the pellet re-suspended in cold FCS at 2-8°C (500 µl per 10 x 106 cells). The Freezing Mix was added so the cells were re-suspended in a solution of FCS to Freezing Mix in a 1:1 ratio, resulting in an approximate cell concentration of 10-12 x 106 cells/ml. 1 mL cell
suspension in freezing medium was dispensed into each cryovial. The cryovials were placed into a freezing container, e.g. Mr Frosty, and then the container was placed in a -80 ˚C freezer. The complete process of cell re-suspension in FM, aliquoting, vial placement into Mr Frosty and transfer of Mr Frosty units into -80˚ C freezer was performed in less than 10 minutes and conducted on ice.
Within 1-3 days at –80 ˚C, the cryovials were transferred from the Mr Frosty units into standard freezer boxes within a liquid nitrogen container.
Thawing Procedure
10mL of TM per sample to be defrosted was pre-warmed in a water bath set at 37°C (± 1°C). One 15 mL falcon tube was labelled per sample. The PBMC cryovial(s) were removed from liquid nitrogen and placed on dry ice until ready to thaw. It was recommended to not thaw more than four cryovials at a time due to time and handling constraints.
Cells were thawed quickly with gentle agitation in a water bath set at 37°C (± 1°C), until a visible ice chunk remained in the cryovial. Each thawed cell suspension was gently transferred to the appropriately labelled 15 mL tube. The vial(s) were rinsed with 1mL TM, adding the TM drop wise to the thawed cells. 10 mL TM was added to each tube slowly and drop-wise; the tubes were swirled gently to mix, thus avoid shear forces. The tubes were centrifuged at room temperature for 5 minutes at 200 x g. The supernatant was discarded and the bottom of the tube was gently tapped to re-suspend the cell pellet. The cell pellet was re-suspended in 1 mL R10 initially, pipetting up and down to get a single cell suspension. Once re-suspended, a further 9 mL R10 was added for a final volume of 10 mL cell suspension. The centrifugation procedure listed above was repeated. The cells were counted using the trypan blue exclusion method. The cells were spun down and re-suspended in sterile R10 to give a final concentration of 4 x 106 cells/mL. The cells were rested at 37°C for 2 hours. The cells were re-counted using trypan blue exclusion after resting and adjusted accordingly to give a final concentration of 4 x 106 cells/mL.
2.2 ELISpot Technique