Click it plus edu alexa flour 647 flow cytometry assay kit

Lentiviral transduction and RA-mediated differentiation of L-AN-5 cells were carried out as described in the section ‘circRNA knockdown and differentiation of L-AN-5 cells’. Labeling of newly synthesized DNA was carried out using Click-iT Plus EdU Alexa Flour 647 Flow Cytometry Assay Kit (Thermo Scientific) according to manufacturer’s protocol. Notably, the cell culture medium of L-AN-5 cells cultured in six-well plates was supplemented with 10 μM EdU for 1.5 hr. To stain total DNA, cells with already detected EdU were resuspended in 400 μl 1x Click-iT saponin-based permeabilization and wash reagent from the Click-iT Plus EdU Alexa Flour 647 Flow Cytometry Assay Kit (Thermo Scientific). Subsequently, RNase A was added to a final concentration of 0.2 mg/ml. After 5 min of incubation at room temperature, propidium iodide was added to a final concentration of 5 μg/ml and the cells were incubated for 30 min at room temperature. Incorporated EdU and total DNA levels were analyzed on a BD LSRFortessa flow cytometer (BD Biosciences). Data analysis was carried out in the FLOWJO software (BD Biosciences). The gating strategy is shown in Figure 2—figure supplement 3A.

For FACS or flow experiments, single-cell suspensions of cells were resuspended in a 2% FBS solution and then stained with appropriate antibodies at a 1:100 dilution for 60 min at 4 °C in the dark. Antibodies used were FITC anti-mouse Ly6d (BioLegend; 138606) and APC anti-human/mouse Cd49f (BioLegend; 313616). Before sorting or flow, SytoxBlue (Thermo Fisher; S34857) was used for a viability dye at a 1:1000 dilution. FACS experiments were run on FACSAria II, and flow experiments were run on LSRII instruments. Both instruments used the BD FACSDiva 8.0.1 software for data collection. For scRNA-Seq experiments, cells were only stained with SytoxBlue, and live cells were sorted for downstream processing and experimentation. For EdU experiments, we used the Click-iT Plus EdU Alexa Flour 647 Flow Cytometry Assay Kit as per manufacturer instructions (Invitrogen; C10634). For Apotracker^TM experiments, we used the Apotracker^TM Green reagent as per manufacturer instructions (BioLegend; 427402). FlowJo 10.6 was used for data analysis.

Approximately 2-5x10⁶ cells from the brain samples or 2x10⁶ splenocytes were transferred to wells of U-bottomed 96-well microtiter plates. For tetramer staining the cells were incubated for 20 min (RT, in the dark) with 50 μl FACS medium (PBS, 1% BSA, 0.1% N_aN₃) containing 10 µL relevant fluorochome labeled tetramers; these were kindly provided by S Buus and A Stryhn (this institute). To prevent unspecific binding cells were blocked with α-CD16/32 and subsequently incubated for 20 min (4°C, in the dark) with 50 µL brilliant violet staining buffer (Biolegend) containing conjugated antibodies (1:100) for the relevant cell-surface markers. Cells to be stained for intracellular granzyme B, T-bet or Eomes expression were permeabilized and stained according to the FoxP3 staining protocol from BD Biosciences. When analyzing the EdU incorporation cells were permeabilized and stained according to the Click-It™ Plus EdU Alexa Flour™ 647 Flow Cytometry Assay Kit from Invitrogen by Thermo Fisher Scientific. Cells were subsequently washed twice in wash media (PBS with 0.1% N_aN₃), resuspended in PBS and stored at 4°C until analysis. The general gating strategy regarding CNS derived cells is depicted in Supplementary Figure 1.