100 μm filter

The animals were sacrificed, and the tail skin was peeled off and cut into 2-3 cm-long pieces. The protocol for the isolation and culture of keratinocytes was based on existing literature (10 (link)). Briefly, the pieces of tail skin were first washed with phosphate-buffered saline (PBS) and incubated overnight with KC growth media (EpiLife^®, Gibco ThermoFisher, NY, USA) containing 4 mg/mL dispase II in a rotator at 4°C. The following day, the pieces of tail skin along with the media were poured into a Petri dish and washed with PBS. The epidermis was lifted using forceps and transferred to a new Petri dish containing trypsin solution at room temperature. The epidermis was then gently agitated using a horizontal shaker at 30 rpm for 20 min. The epidermal tissues were vigorously shaken to disperse the keratinocytes into single cells. The cells were subsequently filtered through a 100 μm filter (Falcon, NY, USA), and centrifuged for 5 min at 180 × g. The supernatants were aspirated, and the pellets were gently resuspended in 1 mL ice-cold KC growth medium. The keratinocytes were seeded in 6-well plates at a density of 1 × 10⁴/cm² in KC growth medium. The medium was replaced after 24 h of initial plating for removing the unattached cells, and the cells were cultured for three days.

Pancreatic tumour cells from Pdx1-Cre-K-MADM-p53 mice were dissociated using a protease cocktail containing collagenase (Worthington), dispase (Roche) and trypsin-EDTA (Invitrogen) in Hank's balanced salt solution, quenched with fetal bovine serum (Hyclone) and passed through a 100 μm filter (Falcon) before plating in DMEM (CellGro) containing 10% fetal bovine serum and penicillin/streptomycin (VWR). Cells were grown in culture for 2–3 days and subject to FACS analysis using a Guava flow cytometry system (Millipore). Stable GFP+/tdTomato− (green) cell lines were obtained through serial passage and confirmed to be p53^KO/KO by PCR genotyping of genomic DNA isolated using QuickExtract (Epicentre). For immunocytochemistry, primary cultured cells were grown on glass coverslips, fixed with PFA, permeabilized with 0.2% Triton X-100, blocked with 5% BSA in PBS and stained with rabbit anti-p53 primary antibody (Novacastra NCL-p53-CM5p, 1:200), donkey anti-rabbit Alexa 647 secondary antibody (Life Technologies A-31573, 1:500) and DAPI before mounting onto glass slides with Vectashield. Unlike with tissue sections, endogenous p53 was readily detectable by immunofluorescence on cultured cells.

Liver specimens were obtained from the margin of the macroscopically tumor-free liver tissue immediately after resection. Samples were taken under sterile conditions, transferred into ice cold (4 °C) William’s E medium, and human primary hepatocytes were immediately isolated under sterile conditions using collagenase. The liver specimen was washed twice with ice-cold HBSS. The washing step was repeated until all blood vessels and connective tissues appeared to have been liberated (usually three washes). The specimens were then cut into small pieces and digested with William’s E medium containing IV collagenase, and incubated at 37 °C for 30 min with gentle shaking at 120 rpm. After the completion of digestion, a final concentration of 10 % (v/v) FBS was added to stop the collagenase reaction. The resulting cell suspension was filtered through a 100-μm filter (BD Falcon) for the removal of solid aggregates. The filtered sample was subsequently centrifuged at 1500 rpm for 5 min at 4 °C. Finally, the cell pellets were washed with HBSS followed by another centrifugation (1500 rpm for 5 min at 4 °C).

The tissue was incubated in Hank’s balanced salt solution (HBSS) with 2500 U potassium penicillin, 2500 μg of streptomycin sulfate, 625 μg of amphotericin B (PSF, Lonza, Basel, Switzerland) for 3 h. Then the mesenteric tissue was cut into small pieces (<1 mm) and incubated in digestion buffer [1.5 mg/mL collagenase I, 1 mg/mL collagenase II and 1% penicillin‒streptomycin in phosphate-buffered saline (PBS) with 1% foetal bovine serum (FBS), all from SIGMA] for 1 h at 37 °C under agitation. The digestion was quenched with DMEM containing 10% FBS, and the dissociated cells were passed through a 100-μM filter (FALCON) and then subjected to centrifugation at 400 × g for 5 min at room temperature (RT). The cell pellet containing the SVF was resuspended in red blood cell lysis buffer for 5 min at RT and then quenched in DMEM containing 10% FBS. After centrifugation, cells were resuspended in HBSS containing 3% FBS and kept on ice for the duration of processing. Cell concentration and viability were determined on a Cellometer Auto 2000 (Nexcelom, USA) after AO/PI staining, and cells with viability of greater than 80% were subjected to 10× Genomics scRNA-seq.

Human AT was weighed and ±0.500 g of tissue was digested using 2 mg/ml Collagenase I (Worthington) in PBS (Invitrogen/sigma) 2% bovine serum albumin (BSA, Sigma), samples were disrupted using an Octolyser (Miltenyi), incubated at 37 °C with intermittent shaking for 45 min, subjected to a second Octolyser dissociation step, ions were chelated by addition of EDTA (0.5 M, Sigma), samples were filtered through a 100-μm filter (BD), and washed with 20 ml of 2% BSA PBS prior to centrifugation at 1700 rpm for 10 min. The cell pellet was resuspended in 2 ml of PBS 2% BSA for flow cytometric analysis.