Facsaria 2 flow sorter

Cell surface proteins were stained with experimentally-titrated fluorophore-conjugated antibodies in MACS buffer (2 % BSA and 2 mM EDTA in PBS) for 30 min on ice. Stained cells were washed then resuspended in MACS buffer for flow cytometric analysis. For intracellular staining, cells were fixed for 20 min on ice with fixation / permeabilization buffer (Invitrogen), washed twice with permeabilization buffer (Invitrogen), stained with experimentally-titrated fluorophore-conjugated antibodies in permeabilization buffer (Invitrogen) for 30 min on ice, then washed and resuspended in MACS buffer. Cytometric analyses were performed using an Attune NxT Focusing Flow Cytometer (Thermo Fisher Scientific), and cell sorting was accomplished with a FACSAria II flow sorter (BD) and cytometry data was analyzed using FlowJo software v10.3 (Treestar, Ashland, OR).

For constitutive overexpression, the coding region of Klf2 was cloned into a modified version of the retroviral expression vector pMSCV (Takara Bio Inc.) which additionally encodes GFP via an IRES site. For inducible overexpression, the vector pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE (Koo et al., 2012 (link)), in which the eGFP cassette has been replaced by Klf2, was used. The coding region of Ascl2 was cloned into pMSCV with a human CD4 reporter instead of GFP. Viral particles were generated in HEK293 cells by calcium phosphate transfection using the packaging plasmids pECO and pCpG. In vitro prestimulated OT-II T cells (OVA_323-339 peptide for 36 h) were infected with retroviral supernatants by centrifugation for 90 min at 700× g, 32°C and 8 µg/ml polybrene. Residual virus was thoroughly washed away before the OT-II cells were used for subsequent studies. Infected cells were either directly transferred into recipient mice or cultured for additional 20 h in vitro to sort for fluorescent protein expressing cells on a FACSAria II flow sorter (BD).