Midimacs separator magnet

PBMC were washed in filter-sterilized PBS, 2 mM EDTA, 0.5% BSA (MACS buffer; 4 °C), and re-suspended in 90 μL of MACS buffer with 10 μL of CD4⁺ T Cell Biotin-Antibody Cocktail (Miltenyi Biotech) per 10⁷ total cells for 5 min at 4 °C. 20 μL of anti-biotin Microbeads (Miltenyi Biotech) per 10⁷ total cells was then added for 10 min at 4 °C, before increasing volume to 500 µl with MACS buffer. The cell suspension was added to a pre-rinsed LD column on a Midi-MACS separator magnet (Miltenyi Biotech). The column was washed with 1 ml of cold MACS buffer and the collected cells added to a second pre-rinsed LD column, this column was washed three times with 1 ml of cold MACS buffer to collect the unlabelled CD4⁺ fraction. CD4⁺ cells were washed and re-suspended in 90 μL of MACS buffer with 10 μL of CD25 Microbeads (Miltenyi Biotech) per 10⁷ total cells for 15 min at 4 °C, before washing in cold MACS buffer and being re-suspended in 500 μL of MACS buffer. The cell suspension was added to a pre-rinsed MS column on a Mini-MACS separator magnet (Miltenyi Biotech), and the column was washed three times with 500 µl of cold MACS buffer to collect the unlabelled CD4⁺ CD25⁻ cell fraction.

Activation beads were constructed using the NK Cell Expansion Kit, as directed by the manufacturer (Miltenyi Biotec). In summary, biotinylated antibodies were bound to paramagnetic beads coated with streptavidin. Antibodies comprised anti-CD2 (Miltenyi Biotec) and anti-DNAM1 (Miltenyi Biotec), either in combination or alone.
RT was accomplished by combining activation beads with 200,000 isolated NK cells at a ratio of 1:2 in 96-well flat-bottom plates in 200 μl 10% X-vivo with 500 U/ml rhIL-2. 48 h later, each co-culture was agitated by repeated pipetting and then transferred to a 1.2-ml microtiter tube (Thermo Fisher Scientific) containing 100 μl of X-vivo 15 medium. To harvest RT beads, the microtiter tube was suspended in the open groove of a horizontally positioned MidiMACS separator magnet (Miltenyi Biotec). After 5 min, the entire volume of the medium was gently extracted from the bottom of the microtiter tube, leaving the beads held by the magnet on the inner sides of the tube. The beads were immediately resuspended by removing them from the magnetic field and adding RPMI-1640. This magnetic isolation process was repeated to further purify the bead fraction. Last, suspensions of beads were transferred to a 1.5-ml tube and stored at 4°C for several days before experimentation.

To sort the iPSC-aCMs from the CFs for CM-specific end-point analysis, the cells were disassociated as described above. Once the cells were in suspension, the PSC-Derived Cardiomyocyte Isolation Kit (Miltenyi Biotec, Gaithersburg, MD) was used to isolate the CMs via negative selection per the manufacturer’s protocol with LS columns and a MidiMACS Separator magnet (Miltenyi Biotec). Only labeling of the non-CM population was performed to reduce handling of the iPSC-aCMs. This has been shown previously to be sufficient for CM isolation (67 (link)) and, in our hands, yielded ~80 to 90% pure iPSC-aCMs and <6% fibroblasts based on cTnT and collagen 1a1 (Col1a1) immunostaining analysis, respectively (fig. S16). CM cell sorting was performed before RT-qPCR, RNA-seq, patch clamping, and Seahorse analysis.