Fix perm cell fixation cell permeabilization kit

Ki‐67 expression was analyzed by flow cytometry. Ki‐67 intracellular staining was performed using FIX & PERM Cell Fixation & Cell Permeabilization Kit (ThermoFisher Scientific Inc., Waltham, MA, USA). Briefly, 24 h after the last nucleofection, 1 × 10⁵ cells were fixed for 15 min at RT in reagent A and subsequently fixed in 100% methanol for 10 min at 4 °C. Then, fixed cells were washed with PBS and permeabilized with reagent B for 30 min at RT with rabbit polyclonal anti‐human Ki‐67 (#ab15580, Abcam) at 1 : 250 dilution. Finally, cells were washed with PBS and incubated with fluorescein isothiocyanate goat anti‐rabbit IgG F(ab0)2 fragment (#F0382, Sigma‐Aldrich, St. Louis, MO, USA) for 30 min at RT. Data acquisition and analysis were performed using a BD FACSCanto II (BD Biosciences). At least 10 000 events were detected for each sample to guarantee a statistical relevance.

For the cell proliferation assays MHC-I-deficient K562-CR2, K562-CR2-HLA-E*0103/0103 or K562-CR2-HLA-E*0101/0101 target cells were established and maintained as described before (16 (link)). The cells were then always infected with the marmoset B-lymphoblastoid cell line B95-8 derived EBV-strain, which encodes for the GGDPHLPTL LMP-1 variant, (MOI=1) (16 (link)) for 3 days. Sorted NKG2C^-NKG2A⁺, NKG2C⁺NKG2A^- and NKG2C⁺NKG2A⁺ NK cells were used as effector cells and were quickly thawed at 37°C, washed, and pre-activated overnight in RPMI, 10% FCS, 1% L-glutamine, 10 ng/ml IL-12 and 100 ng/ml IL-18 at 37°C. The NK cells were then harvested by centrifugation at 400xg for 5 minutes and washed once with Opti-MEM. The NK cells were then cultured together with EBV-infected K562-CR2, K562-CR2-HLA-E*0103/0103 or K562-CR2-HLA-E*0101/0101 cells (E:T, 1:1) in RPMI, 10% FCS, 1% L-glutamine for indicated time points. In some experiments, additional 300 µM of EBV LMP-1 peptides or α-NKG2A blocking monoclonal antibodies (Monalizumab, 10 μg/mL, Innate Pharma) was added after 0, 2, and 4 days, respectively, to the co-culture. All cells were then harvested, fixed with the FIX & PERM Cell Fixation & Cell Permeabilization Kit (Thermo-Scientific) and analysed by flow-cytometry, as described below.

Functional responses of monocytes and macrophages to TLR4 or TLR7/8 ligands were assessed in cell cultures of PBMCs or OptiPrep-isolated IHLs stimulated with either LPS (100 ng/mL, Invivogen) or CL097 (1 μg/mL, Invivogen) in a medium containing BFA (5 μg/mL, Sigma Aldrich), as previously described (29 (link)). Unstimulated cells were used as baseline control. At 16 h post-stimulation, cells were prepared for intracellular cytokine staining following the manufacturer's instructions (FIX & PERM® Cell Fixation & Cell Permeabilization Kit, Thermo Fisher Scientific) and antibodies from Supplementary Table 5. Cells were washed and measured using a BD LSR Fortessa. Further analyses and gating strategies were performed with FlowJo v10 software.

Mice were anesthetized with isoflurane and euthanized by decapitation. Brains were cut in half sagittally and drop-fixed in 4% paraformaldehyde for IHC, and half cerebellum or tumor collected for Drop-Seq. Alternatively, half of the tumor was collected for flow cytometry.
Cells were dissociated using the Papain Dissociation System (Worthington Biochemical) according to the manufacturer’s protocol, as in our prior studies^{50 (link)}. Briefly, tumors or cerebella were dissected from isoflurane-anesthetized C57BL/6 or M-Smo mice and incubated in papain at 37 °C for 15 min. The tissue was then triturated, and the cells were spun down, resuspended and a density gradient was formed with ovomucoid inhibitor. Lastly, cells were resuspended in HBSS with 6 g/L glucose and diluted to ~100 cells/µl for Drop-seq.
Alternatively, cells were resuspended in Hank’s Balanced Salt Solution with 6 g/L glucose, fixed and permeabilized for flow cytometry using the Fix & Perm Cell Fixation & Cell Permeabilization Kit (ThermoFisher Scientific), according to the manufacturer’s protocol. Cells were stained for FxCycle Violet and with GFAP, phospho-RB, and/or MYOD1 as indicated. Samples were run on the Becton Dickinson LSR Fortressa and data were analyzed with FlowJo V10. The gating strategy for excluding debris, doublets and sub-G1 cells is exemplified in Supplementary Fig. 11.