Anti ly 6a e sca 1

Anti-mouse ABs: anti-CD3e (clone: 145-2C11), anti-CD34 (clone: HM34), anti-CD45R (clone: RA3-6B2), anti-CD117 (c-KIT, clone: 2B8), anti-CD182 (CXCR2, clone: SA044G4), anti-CD184 (CXCR4, clone: L276F12), anti-Ly-6A/E (Sca-1, clone: D7), anti-Ki-67 (clone: 11F6), anti-Ly-6G (clone: 1A8), anti-NK-1.1 (clone: PK1), anti-mouse/human-CD11b (clone: M1/70) all from BioLegend (San Diego, CA, USA), and anti-CD16/32 (clone: 93) and anti-CD62L (clone: MEL-14) (both from eBioscience, Thermo Fisher Scientific, Waltham, MA, USA). Anti-human ABs: anti-CD66b (clone: 80H3, Beckman Coulter, Brea, CA, USA), and anti-CD71 (clone: M-A712, BD Biosciences, Franklin Lakes, NJ, USA). Viability dye eFluor780 and eFluor506 (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA) or DAPI (BioLegend, San Diego, CA, USA) were used to determine viable cells.

Stromal Vascular cells (SVC) from the donor site after mechanical stress were isolated as previously described [65 (link)]. Briefly, adipose tissues were digested in phosphate-buffered saline (PBS) with 0.5% BSA and 1 mg/mL type II collagenase for 25 min at 37 °C, and stromal vascular cells (SVC) were separated from adipocytes by centrifugation. Isolated SVC were stained using live/dead fixable dyes (Invitrogen): anti-CD45 (eBioscience, 30-F11), anti-Ly-6A/E(Sca1) (BioLegend, D7, San Diego, CA, USA), anti-CD31 (BioLegend, 390, San Diego, CA, USA), and anti-CD140a (BioLegend, APA5, San Diego, CA, USA) and anti-Ki67 (eBioscience, SolA15, San Diego, CA, USA). Cells were analyzed on a FACS Canto II Flow Cytometer (BD Biosciences, San Jose, CA, USA) using the FlowJo 10.6 software (Tree Star, Ashland, OR, USA).

Enteroid monolayers and 3D organoids were fixed in 4% paraformaldehyde in PBS for 20 minutes followed by a block while permeabilizing in 5% BSA/ 0.2% Triton X-100/ PBS for 30 minutes at room temperature. The stainings were performed overnight at 4°C with the following primary antibodies: anti-Cleaved Caspase-3 (Asp175) (Cell Signaling, #9661), anti-phospho-Histone H3 (Ser10) (Merck-Millipore, #06-570), anti-GFP (Abcam, #ab6673), anti-Phospho-c-Jun (Ser73) (D47G9) (Cell Signaling, #3270), anti-Phospho-JNK1+JNK2 (T183 + Y185) (Abcam, #ab4821) and anti-Ly-6A/E (Sca-1) (Biolegend, cat#108101). Appropriate Alexa Fluor labeled secondary antibodies (ThermoFischer Scientific) were combined with DAPI and/or Phalloidin Alexa Fluor 647 (ThermoFischer Scientific, cat# A22287). For labeling of cells in S-phase, a pulse of 10 μm EdU (5-ethynyl-2′-deoxyuridine) was given one hour prior to fixation and detection was performed according to manufacturer’s guidelines before starting the immunofluorescence staining using Click-iT EdU Cell Proliferation Kit for Imaging Alexa Fluor 647 (ThermoFischer Scientific, cat# C10340).