Dapi 4 6 diamidino 2 phenylindole

Human IgG was purchased from Equitech Bio (Kerrville, TX, USA). Poloxamer-188, RNase-free water, 4,6-diamidino-2-phenylindole (DAPI), and fetal bovine albumin (FBS) were obtained from Fisher Scientific. Antineutotensin receptor 1-monoclonal antiboby (anti-NTSR1-mAb was purchased from Santa Cruz Biotechnologies. siRNA against mutated KRAS G12S was designed andpurchased from GE Dharmacon. siG12S sense and antisense sequences are GUUGGAGCUAGUGGCGUAGdTdT and CUACGCCACUAGCUCCAACdTdT respectively. siGLO-Green (6-FAM-labeled) was obtained from GE Dharmacon. N-[p-maleimidophenyl] isocyanate (PMPI), and 2-iminothiolane-HCl were obtained form Thermo Scientific. Neurotensin was purchased from Abcam (Cambridge, MA, USA)

Cellular composition of the liquid cultures and in vivo tissues were analyzed using flow cytometry. The following antibodies and recombinant proteins were used: Sca1-Pacific blue (PB), CD34-Phycoerythrin (PE), CD48-PE, CD150-PE.Cy7, Ter119 Allophycocyanin (APC)(all Biolegend), CD117-APC, CD16/32-APC.Cy7, CD11b-PE, Gr1-APC, CD3-PE.Cy7, CD71 PE (all BD Biosciences), CD19 Alexa Fluor 700 (AF700, Life) and CD117 PerCpCy5.5 (SONY). Lineage positive cells were detected using the murine lineage detection kit (BD Biosciences) subsequently stained with streptavidin-pacific orange (PO, Fisher Scientific).
SSEA4-PE (Biolegend) and Tra-1-60 Alexa Fluor 647 (AF647, BD Biosciences) were used for regular pluripotency screenings of the iPSCs. Hematopoietic induction of iPSC was assessed using CD34-PE and CD45-BV421 antibodies (both BD Biosciences). Dead cells were excluded by using 7-aminoactinomycin-D (7AAD, Life) or 4’,6-Diamidino-2-Phenylindole (DAPI, Fisher Scientific). Flow cytometry was performed using a BD LSRII flow cytometer (BD Biosciences). For subsequent cell sorting a FACSAria (BD Biosciences) was used. Analysis of FACS data was performed using FlowJo (TreeStar).

Caco-2 cells were plated in 96-well collagen IV–coated plates at 1.5 × 10⁴ cells and cultured for 48 hours. Cells were treated with 250 μM indomethacin or vehicle control (DMSO) for 16 hours overnight. The next day, cells were intoxicated with recombinant TcdB at a concentration of 50 nM for 4 hours. After infection, cells were washed twice with PBS and fixed by incubation with 4% paraformaldehyde for 20 min. Cells were washed with PBS and permeabilized with 0.1% Triton X-100 (MP Biomedicals) for 15 min. To block nonspecific binding, cells were incubated with 10% goat serum (Fisher Scientific) for an hour at room temperature, followed by staining with primary anti–ZO-1 mouse monoclonal antibody (BD Biosciences) at a concentration of 25 mg/ml overnight at 4°C. The next day, cells were washed with 1% goat serum twice for 10 min, followed by incubation with secondary anti-mouse Alexa Fluor 488 antibody (Thermo Fisher Scientific) at a concentration of 8 μg/ml for 2 hours at room temperature. Cells were washed twice with 1% goat serum, followed by staining with 4′,6-diamidino-2-phenylindole (DAPI; Fisher Scientific) at a concentration of 300 ng/ml for 10 min at room temperature. Cells were imaged in an Evos FL Auto using the green fluorescent protein and DAPI filters at a ×20 magnification.