DMSO cryopreservation

Cells were cryopreserved using a modified version of the protocol described by 10x Genomics (CG00039, Rev C). Briefly, cell suspensions were centrifuged for 5 min at 300 g and 4 °C. Supernatants were discarded and the cell pellets were resuspended in pre-chilled (4 °C) DMSO/FCS solution containing 10% of DMSO. Aliquots of 1 mL were dispensed into cryovials and placed into a CoolCell^® (BioCision, Larkspur, CA) that was pre-cooled in the fridge at 4 °C for at least two hours. The CoolCell^® was stored overnight at −80 °C and the cryovials were then transferred to −150 °C storage. Aliquots contained 4.5 × 10⁶, 3.2 × 10⁶ and between 2.1 × 10⁶ and 8.5 × 10⁴ cells for the species mixing experiment, MDMs and rat liver immune cells, respectively.

Frozen samples were prepared for scRNA-seq by rapidly thawing them at 37 °C and transferring the cells into 50 mL centrifuge tubes. The cryovials were rinsed with 1 mL of warm (37 °C) medium which was then added dropwise to the DMSO containing fraction while gently shaking the cells. DMEM was used for the species mixing experiment while RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA) supplemented with 10% of FCS was used for primary and primary-derived cells. Next, the cells were gradually diluted by first adding 2 mL of medium followed by another 4, 8 and 16 mL respectively. The cell suspension was gently swirled for 5 sec and incubated for 1 min in between the four dilution steps. The diluted cell suspension was centrifuged for 5 min at 300 g and most of the medium was discarded leaving approximately 1 mL of supernatant. The cells were gently resuspended in the remaining supernatant and washed by adding 9 mL of medium. After centrifugation for 5 min at 300 g the supernatant was discarded and the cells were washed in 1.5 mL of PBS/BSA buffer. Finally, the cells were resuspended in PBS/BSA buffer, filtered through a 40 µm cell strainer and stored on ice until counting and loading of the cells into the scRNA-seq device. For immune cells HBSS was used instead of PBS.