After density gradient centrifugation, different blood cells are found in different layers according to their density. From bottom to top you find red blood cells and granulocytes, followed by the density gradient medium (e.g. Lymphoprep), the layer containing the PBMCs and on top the plasma phase containing platelets
Centrifuge for 10 min at 150 × g at 4 °C with the centrifuge brake switched off.
Decant the supernatant, resuspend the pellet in RPMI medium and centrifuge again for 10 min at 150 × g and 4 °C with the centrifuge brake off (see Note 10 ).
Decant the supernatant and resuspend the resulting pellet in cell medium (see Note 11 ).
Count the cells (see Note 12 ).
Freeze the cells or perform an assay with the freshly isolated cells.
3.2 Cryopreservation of PBMCs
Prepare cryovials and label them with cryotags or with a permanent marker (see Note 13 ).
Define the concentration at which you want to freeze the PBMCs and the quantity of cells contained in one aliquot. Bring the cell suspension to the double concentration of your end concentration by adding cell medium.
Add an equal volume of freezing medium in three subsequent steps in intervals of 3–4 min to give your end concentration (see Note 14 ).
Mix the cell suspension and transfer aliquots of the PBMCs to the cryovials.
Freeze the cells in a −80 °C freezer as fast as possible.
Transfer the cells to liquid nitrogen or to a freezer providing temperatures lower than −150 °C within 2–3 days. Store them at this low temperature until use.
3.3 Thawing PBMCs
Warm RPMI medium in a water bath at 37 °C for washing your thawed cells in (see Note 15 ).
Thaw the cells in the water bath (see Note 16 ).
Quickly resuspend the cells in the medium and centrifuge at 150 × g for 10 min at 4 °C with the centrifuge brake off.
Decant the supernatant, resuspend the cells in RPMI and wash again as in step 3 (see Note 17 ).
Resuspend the cells in cell medium and count them.
3.4 Culturing Cells
3.5 Stimulating Cells
When you perform an assay with PBMCs and stimulate them with different antigens, always introduce positive and negative control wells (see Note 22 ).
Pre-establish the optimal concentrations of your stimuli in titration experiments.
After the incubation time of your assay has ended, collect the supernatant and freeze it at −80 °C. You can later perform multiplex analyses to determine the prevalent cytokine milieu in different stimulatory situations.
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It can be highly recommended to use one-way plastic pipettes in immunological settings. Lipopolysaccharide, when sticking to glass pipettes, can withstand autoclaving and stimulate cells coming into contact with it.
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