Earlier studies have examined DNA methylation profiles in PBMCs in human diseases (Li et al., 2010b; Steegenga et al., 2014). However, any disease-related signatures identified would just display differences in the relative abundance of individual cell types as each cell subset generates a unique methylation profile (Reinius et al., 2012). Therefore it is important to isolate specific cell types to identify cell- or tissue-specific changes in DNA methylation to avoid confounding variables. This method involved isolating CD19+ B cells, CD14+CD16- monocytes and CD4+ T cells for epigenetic profiling. The classical CD14+CD16- monocytes were studied instead of CD14+ cells as in the preliminary study to remove proinflammatory macrophages (Ziegler-Heitbrock, 2007).
2.4.1 B cell isolation
Blood taken in heparin vacutainers was diluted 1:1 with Roswell Park Memorial Institute (RPMI) and left on the roller overnight. The diluted blood was carefully layered onto 12.5 mL Percoll 1.078g/mL in 50 mL Falcon tubes and centrifuged at 800 xg for 20 minutes with no brake, at 20°C. Percoll was used as it is a simple method for isolating PBMCs (de Almeida et al., 2000). The sample had separated into different layers (Figure 2.1) where most of the plasma layer was removed before the PBMC ring was transferred to new 50 mL Falcon tubes. The tubes were topped up with phosphate buffered saline (PBS) and ethylenediaminetetraacetic acid (EDTA) and spun at 550 xg for eight minutes at 20°C. The cell pellets were
did not significantly affect gene expression (Lyons et al., 2007). The cell pellet was resuspended in 80 µL PBS and EDTA per 10 million cells. Then 20 µL of CD19 microbeads was added per 10 million cells. This was left to incubate for 15 minutes at 4°C. The samples were washed with 2 mL of PBS and EDTA and spun at 300 xg for six minutes at 4°C. Whilst the samples were spinning, an LS column was washed with 3 mL PBS and EDTA through a pre-separation filter. The cells were resuspended in 500 µL of PBS and EDTA and applied to the column through the pre-separation filter. The flow-through was collected in another tube placed under the column. 3 mL of PBS and EDTA was added to the tubes to collect any remaining cells and added to the column three more times. The column was removed from the magnet and 5 mL of PBS and EDTA was added to the column. The purified cells were flushed out using the plunger. The flow-through and purified cells were spun at 300 xg for six minutes at 4°C. The purified cells were applied to another column to order to increase the purity. Whilst the cells were spinning, an MS column was washed with 500 µL of PBS and EDTA. The supernatant from the CD19- tube was decanted and left on ice to one side. The CD19+ fraction was resuspended in 500 µL of PBS and EDTA and added to the MS column. 500 µL of PBS and EDTA was added to the tubes and then to the column three more times. The column was removed from the magnet and 2 mL of PBS and EDTA was added to the column to flush out the purified cells. The flow-through was discarded and purified cells counted.
Plasma PBMC layer Percoll
2.4.2 Monocyte isolation
The cell pellet left to one side from Section 2.4.1 (first CD19- fraction) was resuspended in 50 µL PBS and EDTA per 50 million cells. 50 µL of CD16 microbeads was added per 50 million cells. The tubes were incubated for 30 minutes at 4°C and washed by adding 2 mL of PBS and EDTA and then spun at 300 xg for six minutes at 4°C. Whilst the cells were spinning, an LD column was washed with 2 mL PBS and EDTA. The cell pellet was resuspended in 500 µL of PBS and EDTA and applied to the column. 1 mL of PBS and EDTA was added to the tubes to collect any remaining cells and added to column twice more. The flow-through was collected and a cell count was performed. After depletion of the CD16+ cells, the sample was spun at 300 xg for six minutes at 4°C. The pellet was resuspended in 80 µL PBS and EDTA per 10 million cells. 20 µL of CD14 microbeads was added per 10 million cells and incubated for 15 minutes at 4°C. The cells were washed with 2 mL of PBS and EDTA and spun at 300 xg for six minutes at 4°C. Whilst the cells were spinning, an MS column was washed with 500 µL PBS and EDTA. The cells were resuspended in 500 µL of PBS and EDTA and applied to the column. 500 µL of PBS and EDTA was added to the tubes and then the column three times. The column was removed from the magnet and 2 mL of PBS and EDTA was added to flush out the purified cells. The flow-through was collected, counted and spun at 300 xg for six minutes at 4°C.
2.4.3 T cell isolation
For CD4+ T cell isolation, the cell pellet from Section 2.4.2 was resuspended in 80 µL PBS and EDTA per 10 million cells. 20 µL of CD4 microbeads was added per 10 million cells. The tubes were incubated for 15 minutes at 4°C and washed with 2 mL of PBS and EDTA and spun at 300 xg for six minutes at 4°C. Whilst the cells were spinning, an MS column was washed with 500 µL PBS and EDTA. The cells were resuspended in 500 µL of PBS and EDTA and applied to the column. 500 µL of PBS and EDTA was added to the tubes and then to the column three times. The column was removed from the magnet and 2 mL of PBS and EDTA was added to the
200 µL PBS and 200 µL Buffer AL from the QIAamp DNA Blood Mini Kit, Qiagen, vortexed and stored at -20°C for later DNA extraction. The other pellet was resuspended in 300 µL RNAProtect, vortexed and stored at -80°C for later RNA extraction.
2.4.4 Optimisation of the immune cell isolation protocol
Initially, PBMCs extracted from the samples were split into three to try and positively select the three different cell types. However, this resulted in low cell numbers and therefore would not obtain enough DNA for downstream experiments. The harvested PBMCs were then split into two, one to sort for CD19+ only and the other to sort for CD14+CD16- and then CD4+ cells. However, this also resulted in low cell numbers for CD19+. As CD19+ cells make up only 5-15% of lymphocytes (Fettke et al., 2014), it was crucial to start with all the PBMCs.
Additionally, a second Percoll step was included in Section 2.4.1 to increase the purity of the cells and to reduce debris in the samples. However, this step was later removed as final cell numbers were low and subsequently, resulted in low DNA concentrations. The purity of CD19+ cells dropped significantly, hence the addition of another column step for this cell type to increase the purity to over 95%.