PIEZO1 activation and signalling is associated with inflammation permissive phenocopy of synovial resident macrophages in early arthritis
PIEZO1 activation and signalling is associated with inflammation permissive phenocopy of synovial resident macrophages in early arthritis
On the days of the extraction, the cells were washed with D-PBS, which was collected in individual tubes. Then the cells were lifted using Accutase (Thermoo, 00-4555-56) for 10 minutes at 37oC. The cell suspension was then transferred into the respective tubes, and the wells were washed once with D-PBS to collect all the cells. The cells were spun for 400g for 8 minutes, and resuspended in 1 mL of FACS buffer. The cells were kept on ice, and cell numbers were counted. For each condition (12 in total), 3500 cells were isolated into low-bind Eppendorf tubes.   The cells were then tagged in FACS buffer using the BD Human multiplex tags (BD, 633781), 5uL of Tags/50ul of cell suspension. After  20 minutes incubation, the cells were washed three times, pooled together, and loaded onto the scRNA-seq BD Rhapsody Cartridge of single-cell RNA extraction, using the BD Rhapsody Cartridge Reagent Kit (no. 633731) according to the manufacturer's protocol.   Macrophage 72hour stimulation with Yoda1  Monocyte-derived macrophages were cultured in media supplemented with 20 ng/mL MCSF for 3 days, and then stimulated with Yoda1 20 uM for another three days before scRNA extraction.  In macrophage cultures, the media was replenished at day 3 with new M-CSF supplementation. In all experiments, cells cultured with equivalent DMSO or only in M-CSF supplemented medium were used as controls. The volume of media was adjusted so that the hydrostatic pressure was 103Pa in all experiments.   Overnight PIEZO1 activation using Yoda1  Macrophages were differentiated from CD14 monocytes as described above for 6 days in complete RPMI supplemented with 20 ng/mL M-CSF, changing medium on day 3. On day 5 of differentiation, the cells were stimulated with 20 uM Yoda1, for 12 hours (overnight).   Both complete removal of media, or the exiting media were tested. In metadata this is shows as \"change\", where the whole media was removed and new media added with Yoda1, and \"No_change\", in which the existing media was directly supplemented.  Fibroblasts and Macrophage cocultures   Synovial fibroblasts were seeded in 24 well plates at a density of 98x10^3 cells/well. The cells were allowed to grow for 72 hours in complete DMEM (Invitrogen 41965039), with 10% FBS (Sigma, F9665), 1% non-essential amino acid (Thermofisher 11140035), sodium pyruvate (Invitrogen 41965039). Simultaneously, macrophages from different donors were extracted, and grown in 6 well plate in complete RPMI medium with 50 ng/mL M-CSF. For setting up cocultures, the macrophages were lifted using Accutase as described earlier, and added onto the confluent FLS monolayers in complete RPMI, at starting seeding densities 1:1.   72hours stimulation of cocultures To stimulate the cocultures for 72 hours, macrophages were allowed to attach to the FLS monolayer for at least 6 hours. Then without changing medium, Yoda1 or equivalent DMSO controls were added in complete RPMI (2ml), to a working concentration of 5uM. The cocultures were cultures like this for 72 hours until extraction for single-cell RNA sequencing. Overnight Stimulation of cocultures  To stimulate the cocultures overnight, macrophages were allowed to attach to the FLS and were cultured together for 60 hours. Then Yoda1 or equivalent DMSO controls were added in complete RPMI (2ml), to a working concentration of 5uM. The cocultures were cultures like this for 12 hours until extraction for single-cell RNA sequencing.  The cells were tagged in FACS buffer using the BD Human multiplex tags (BD, 633781), each tag representing a condition/donor. After cell tagging, the cells were washed three times, pooled together, and loaded onto the scRNA-seq BD Rhapsody Cartridge of single-cell RNA extraction, using the BD Rhapsody Cartridge Reagent Kit (no. 633731) according to the manufacturer's protocol.  cDNA was synthesised on mRNA captured on the beads using BD Rhapsody cDNA Kit (Cat. No. 633773), following manufacture's protocol.   Whole transcriptome libraries were prepared using the Whole transcriptome Analysis and Sample Tag library Preparation kit (BD 633801), as per manufacture's protocol (2019 version).  