Detection of bacteria-residing circulating tumor cells (CTCs) using DARMR

Fresh blood samples (3–5 mL) from cancer patients were collected into EDTA-coated tubes and immediately labeled with 10 μM DARMR, incubated at 37°C for 0.5 h. Red blood cells were lysed twice by two volumes of ACK lysis buffer (C3702-500mL, Beyotime biotechnology) on ice for 5 min. The pellet was resuspended in PBS, and CTC isolation was performed using a size-dictated immunocapture chip (SDI-Chip) as described previously.The SDI-Chip was fabricated using PDMS through soft lithography, with SU8-3050 photoresist (Alfa Chemistry #SU8-3050) for micropillar mold preparation. Surface modification involved 4% MPTS (3-mercaptopropyl trimethoxysilane; Sigma-Aldrich #175617-100G), 0.01 μmol/mL GMBS (N-γ-maleimidobutyryl-oxysuccinimide ester; Sigma-Aldrich #442630-100MG), and 20 μg/mL biotinylated anti-EpCAM antibody immobilized via streptavidin (Sigma-Aldrich #S4762). Nonspecific binding was blocked with 3% BSA /0.05% Tween 20 in PBS.Samples were introduced into the chip at 1 mL/h using a syringe pump, and captured cells were released with 0.25% trypsin at 37°C for 10 min. Viability was assessed by Calcein-AM/PI (Solarbio #CA1630), and immunostaining used Mouse anti-human Cytokeratin Antibody anti-human FITC (1:200, Miltenyi #130-118-964), Rabbit anti-human CD68 Alexa Fluor® 647 (1:200, Biolegend #333820), mouse anti-human CD45 APC (1:200, BD Biosciences #555485), and DAPI (1:1000, ThermoFisher Scientific #D1306) for nuclear counters taining. Images were acquired on a Zeiss LSM 900 confocal microscope (Carl Zeiss, Germany) equipped with Airyscan 2 super-resolution detector using a 10×/0.45 NA Plan-Apochromat objective.