Lsrfortessa x50

Single cell suspensions were prepared and stained for with the relevant antibodies. BRDU staining was performed following the fixation/permeabilization reagents from the BD BrdU flow kit according to manufacturer’s instructions but using the Mobu-1 antibody clone (Thermo Fisher Scientific). Intranuclear BCL6 staining was performed on cells permeabilised using the eBioscience Foxp3 / Transcription Factor Staining Buffer Set (Invitrogen). Active caspase 3 staining was performed on cells permeabilised using the Fixation/Permeabilization Solution Kit (BD bioscience). All permeablization steps were performed with an overnight incubation at 4°C. Samples were analyzed using BD LSR Fortessa, LSR Fortessa x20, LSR Fortessa x50, LSR II, or Attune NxT machines. Analysis was performed with Flowjo (Treestar Inc.). Single cell index sorting for sequencing and cell culture experiments were performed using the BD FACs Aria II, FACs Aria II SORP, or Fusion II machines. FACs sorts were performed using 85uM or 100uM (when culturing) nozzles.

Analysis of transduced cells was performed using an LSRFortessa X-50 (BD) at the FHCC Flow Cytometry Core Facility. 150,000 lenti-X 293T cells were infected with serially diluted concentrated lentivirus; one well per plate had its cells counted to calculate an accurate initial cell number. To infect cells, 500 μl diluted viral supernatant was used per well. Cells were left for 48 h before FACS analysis. The cells were then washed gently with PBS and trypsinized with TrypLE into single cells. The cells were then collected and filtered through 35 μm FACS tube filters (Corning; 352235). 1.0 μg/ml DAPI was added to stain dead cells before proceeding to FACS analysis. Duplicate samples were performed per virus dilution. Titre, calculated as transducing units per ml (TU/ml), was calculated using samples whose florescence level in living single cells was above 1% and below 20%, in order to generate a titre from within the linear and detectable range.
The concentrated virus used for microinjections was found to be within the same order of magnitude of concentration: pSGEP-luci = 9.2 × 10⁷ TU/ml; pSGEP-p53_843 = 1.3 × 10⁸ TU/ml; pSGEP-p53_1558 = 6.5 × 10⁷ TU/ml; and H2B-RFP = 7.1 × 10⁷ TU/ml. This finding is in line with the fact they were produced at the same time using an identical protocol (see above).

Cells were struck out in YES, grown in liquid YES overnight to saturation, and then diluted 1:15 before further growth in liquid YES up to midlog phase. Flow analysis was performed on an LSR Fortessa X50 (BD Biosciences) and color compensation, analysis, and plotting were performed as described previously (Greenstein et al. 2022 (link)).

Flow cytometry was performed on a LSRII (BD Biosciences), LSRFortessa X-50 (BD Biosciences), or NovoCyte 2060R (Agilent) cytometer and analyzed using NovoExpress v1.3.0 (RRID:SCR_024676; Agilent) or Kaluza v2.1 (RRID:SCR_016182; BD Biosciences).