Cytoflex srt

RAW264.7 macrophage cells were infected with stationary phase L. donovani DD8-GFP promastigotes at 1: 10 ratio in DMEM media with 2% FBS. After 2 h incubation the cells were washed with 1× PBS to remove non-internalized parasites and infected cells were sorted based on green fluorescence of GFP in CytoFLEX SRT (Beckman Coulter). The sorted cells were seeded in 6-well plates for an additional 24 h with fresh complete media to convert promastigotes to amastigotes and thereafter treated with 10 μM NCL for 6 h, 24 h, and 48 h, and microscopic images were taken. The percentage of infected macrophages was obtained by flow cytometry analysis.

Cardiac cell suspensions were prepared as described with modifications 17 (link), 18 (link). In brief, mouse hearts were removed, cut into small pieces, and digested with 2 mg/mL collagenase II and 0.5 mg/mL dispase II for 30 min at 37 °C. The mixture was filtered with a 100 μm filter to remove tissue blocks in the cell dissociation solution. The mixture was then centrifuged at 1000 × g for 5 min, and was resuspended with PBS. Cardiac cells were sorted using flow cytometry (Beckman CytoFLEX SRT, Brea, CA, USA) into populations of CD45^- PDGFR-α^- CD31^- cardiomyocytes, CD45^- PDGFR-α⁺ CFs, CD45^- CD31⁺ endothelial cells, and CD45⁺ F4/80⁺ macrophages. Isolated cells used for RNA extraction and q-PCR analysis.

No cell lines used in this study were found in the database of commonly misidentified cell lines (maintained by ICLAC and NCBI Biosample). Ins1E cells were regularly tested negative for mycoplasma contamination (Eurofins) and their identity confirmed by functional insulin secretion assays, qPCR, immunofluorescence stainings and western blots for β-cell marker mRNAs/proteins. Ins1E cells (including knockout cell lines) were cultivated in RPMI 1640 with 10% FCS, 2 mM Glutamax, 10 mM HEPES, 2 mM Na-pyruvate, 0.18 mM 2-mercaptoethanol and 100 U ml⁻¹ penicillin–streptomycin (all Thermo Fisher). CRISPR knockout lines were engineered according to a double single guide RNA (sgRNA) strategy²⁹ using the PX459 plasmid or a modified PX458 plasmid^{55 (link)}. pSpCas9(BB)−2A-GFP (PX458) and pSpCas9(BB)−2A-Puro (PX459) were a gift from Feng Zhang (Addgene plasmids #48138 and #48139). Positive clones were enriched using either puromycin selection or FACS sorting using a FACSAria III (BD Biosciences, running FACSDiva software) or a Cytoflex SRT (Beckman Coulter, running CytExpert software). Monoclonal colonies were grown until near confluency in a 96-well plate, and after passaging, knockout was confirmed using deletion PCR, qPCR and/or immunoblot. At least three monoclonal cell lines were pooled before experiments^{56 (link)}, if not denoted otherwise in the figure legends.

Primary mouse hepatocytes were isolated by the previously reported method with minor modification.^[46] Briefly, the WT and LKO mice were anesthetized with Avertin (Sigma–Aldrich, T48402, 240 mg kg⁻¹) by intraperitoneal injection, and the hepatic portal vein was cannulated. The liver was perfused at 6 mL min⁻¹ with pre‐warmed perfusion medium containing EGTA for 8 min. After the first wash, a second perfusion was performed with prewarmed digestion medium containing collagenase IV at 4 mL min⁻¹ for 5 min. Hepatocytes were filtered through a 100 mm filter membrane, separated by centrifugation at low speed (50 g, 5 min), and purified with Percoll (Sigma–Aldrich, P4937) gradient centrifugation. For cell sorting, Isolated hepatocytes sent to FACS, sorting was performed at 4 °C and samples were collected in growth media (high‐glucose DMEM supplemented with 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin). Only GFP‐positive cells were sorted with Cytoflex SRT (Beckman Coulter, Pasadena, CA, USA) using a 100‐mm nozzle. Sorted cells were immediately centrifuged and resuspended in RNAiso Plus (Takara, 9018) for total RNA isolation or RIPA lysis buffer (Beyotime, P0013B) for immunoblotting assay.

CEACAM5-positive colorectal, lung, or gastric cancer cell lines were purchased from ATCC (SK-CO-1, SNU-C1, H508, LS174T, SNU-16, H727, H2122) or DSMZ (MKN-45), and cultured according to the respective datasheets. CEACAM5-negative A549 cells (ATCC), and two primary epithelial cell lines, i.e., HBEpiC (ScienCell Research Laboratories) and CCD841CoN (ATCC), were used as controls. The number of CEACAM5 and CD47 antigens/cell was determined by QIFIKIT (Agilent DAKO) according to the manufacturer’s instruction as previously described (41 (link)).
MC38 (Kerafast) were silenced for mouse CD47 cell surface expression and were afterwards stably transfected to express human CD47 and human CEACAM5. A stable clone named 3C12 was isolated by cell sorting (Beckman #Cytoflex SRT) and cultured like mentioned in the original MC38 datasheet. The number of human CEACAM5 (203’000 molecules/cell) and human CD47 (40’000 molecules/cell) expressed at the surface of the clone was determined by QIFIKIT (Agilent DAKO) according to the manufacturer’s instruction as previously described (41 (link)).

The BMDM was seeded in 35 mm‐confocal dishes at the density of 2 × 105 cells with complete medium containing nothing or 100 ng ml⁻¹ LPS and 50 ng ml⁻¹ INF‐γ or 10 ng ml⁻¹ IL‐4 for 24 h. The complete medium was then replaced with complete medium containing 10% exosome‐depleted FBS and incubated with 1 × 10⁸ AnCar‐Exo^LaIMTS3 for 12 h at 37 °C. Later, Collected the Mφ, pro‐inflammatory and anti‐inflammatory macrophages and resuspended them with FACS buffer (PBS containing 1% FBS, 1 mM EDTA). Next, prepared for blank control, and Isotype control for the following analysis. Incubated with antibodies against F4/80, CD86, CD206 (1:100, Biolegend) at 4 °C for 30 min. Then, washed with FACS buffer. The cells were centrifuged at 500 g for 5 min. At last, over 50 000 events were collected for analyzing by Flow cytometry (Beckman, CytoFLEX SRT).