Annexin 5 af647

A total of 100,000 control or BCL11A knockdown cells (in triplicates) were seeded in six-well plates and allowed to recover for 48 h. Cell were then collected and stained using the Annexin-V-AF647 (BioLegend) following the manufacturer’s instructions, and cells were then quantified using fluorescence-activated cell sorting.

TRAC KO or electroporation only negative control IGRP_265-273 reactive CD8⁺ T cells were assayed for their capacity to kill the HLA-A*02:01-expressing human β-cell line βLox5, as previously described (9 (link)). In brief, βLox5 cells were harvested from flasks by incubating with Cell Dissociation Buffer for 5-10 minutes at 37°C and 5% CO₂ (Gibco). βLox5 cells were then washed, resuspended at 5-10 x 10⁶ /mL in PBS, and labeled with 5uM CellTrace Violet (Thermo Fisher Scientific) according to manufacturer’s instructions. Labeled βLox5 were plated at a concentration of 30,000 cells per well in a 48-well tissue culture treated plate in complete DMEM (cDMEM) formulated as previously described (27 (link)). After 24hr, CD8⁺ T cell avatars were plated with βLox5 at 0:1, 1:1, and 5:1 effector:target (E:T) ratios in duplicate, and co-cultured for 16 hours at 37°C and 5% CO₂. Cells were harvested and stained with a master mix comprised of 88uL Annexin V-Binding Buffer (BioLegend), 2uL Annexin V-AF647 (BioLegend) and 10uL of propidium iodide at 3mg/mL (PI, Invitrogen). Data were acquired on an LSRFortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software v10 (BD).

Single cell suspensions were washed (2% FCS in PBS), and Fc receptors were blocked with aCD16/CD32 (clone 2.4G2 clone; Biolegend, San Diego, CA, USA) for 10 min. Afterwards, cells were incubated with receptor-specific antibodies (Table S1) obtained from ThermoFisher (Waltham, MA) or BD Biosciences (Franklin Lakes, NJ, USA) for 20 min at 4 °C. Afterwards, cell viability was assessed by using fixable viability dye (FVD)-450 and -780, 7AAD, Sytox AADvanced (all from ThermoFisher) and Annexin V-AF647 (Biolegend), as recommended by the manufacturer. Samples were subjected to flow cytometry using a Attune NxT Flow Cytometer (Thermo Scientific) and an LSRII (BD Biosciences), and analyzed with Attune NxT ((ThermoScientific and FlowJo software, respectively (BD Biosciences)). The gating strategies for liver NPCs (see Figure S1) and splenic leukocytes (see Figure S2A) are depicted in the supplementals.

All fluorescently labeled antibodies were purchased from BD Biosciences, BioLegend and Invitrogen (Thermo Fisher Scientific) (S1 Table). Lysis of erythrocytes in whole blood was done with NH₄Cl. Spleens were mechanically disrupted and filtered through a 70 μm cell strainer before separation of mononuclear cells on Ficoll-Paque gradients. Cell suspensions were stained with antibodies for 30 min on ice, washed, and analyzed on FACSCanto or LSR Fortessa cytometers (BD Biosciences). Analysis of flow cytometric data was performed with FlowJo (Tree Star). The absolute numbers of leukocytes in each category were computed from the white blood cell count measured with a Beckman Coulter AcT diff Analyzer. Early apoptotic cells were assessed by incubating cells with AF647 annexin V (BioLegend) and 7AAD (BioLegend) diluted in annexin binding buffer for 15 min at room temperature in the dark, 400 μl binding buffer was added and the staining was directly analyzed on FACSCanto.