Moflo xdp high speed sorter

CD11b⁺ cells were FACS sorted from naïve murine livers, or those bearing HT29 metastases using the MoFlo XDP high-speed sorter (Beckman Coulter) and cultured for 24 h in serum-free media. The conditioned supernatant was centrifuged at 1500 rpm to remove any cellular debris. Proteome profiler membranes were incubated in the conditioned media and the assay was performed as per the manufacturer’s instructions (ARY028, R&D Systems, Minneapolis, MN, USA). Membranes were developed using the Odyssey Fc imaging system (LI-COR Biosciences, Lincoln, NE, USA) and the intensity was determined using in-house software with normalisation to positive control.

Cells of the promoter library transformants were grown overnight, diluted in 4 mL of liquid SD-HIS medium to an initial OD₆₀₀ ≈ 0.1, and cultured for 5 h at 30℃ and 250 rpm. The cells were collected by centrifugation at 6,000 g for 5 min, washed twice with phosphate-buffered saline (PBS), and finally diluted with PBS to an OD₆₀₀ of 0.1 before flow cytometry. GFP-positive cells isolated were isolated via FACS (excitation 488 nm, emission peak 507 nm) using a MoFlo XDP high-speed sorter (Beckman Coulter, Fullerton, CA, USA). A minimum of 60,000 total events per sample tube was collected for analysis at a flow rate of 2000 events per second. Flow cytometry data were analyzed using Summit software (version 5.2). After sorting approximately 3.7 million library cells, 8,000 cells with the strongest fluorescence intensity were isolated and plated onto SD-HIS agar plates to obtain single colonies. Colonies were randomly picked and cultured in liquid SD-HIS medium using a 96-deep well plate for 24 h. Then, 200 μL cultures were transferred into a 96-deep microplate to analyze GFP expression by measuring the fluorescence intensity using a plate reader as described above.