PBMCs were isolated from peripheral blood samples by density gradient centrifugation with Pancoll Human (PAN-Biotech, Cat. No. P04-60100). Serum samples were obtained by centrifugation in designated containers. All blood samples were taken prior to surgery. Tumor and mucosa samples were taken during tumor biopsy or surgical resection and immediately processed. Unfixed tissue was mechanically and enzymatically (100U/mL DNase, Applichem; Cat. No. A3778 and 320U/mL Collagenase IV, Worthington; Cat. No. LS004180) dissolved using a gentleMACS Dissociator (Miltenyi). Cells were transferred into single cell suspensions for flow cytometry by filtering through 100µm and 70µm nylon cell strainers (Greiner Bio-One; Cat. No. 89508–344 and 89508–340).
Nylon cell strainer
Manufactured by Greiner
Nylon cell strainers are laboratory equipment used to separate cells from a cell suspension. They feature a fine-mesh nylon screen that allows the passage of fluid and small particles while retaining larger cells.
Automatically generated - may contain errors
4 protocols using nylon cell strainer
1
Isolation and Dissociation of PBMCs and Tissue Samples
2
Isolation of PBMCs and Tumor Cells
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Peripheral blood was obtained from patients and age- and sex-matched healthy donors immediately prior to surgery. PBMC were purified using density-based separation with Pancoll Human (PAN-Biotech, Cat. No. P04-60100). Fresh unfixed tissue from primary tumors or non-cancerous mucosa, which was not required for pathological analyses, was processed immediately after tumor biopsy or surgical resection. Fresh tissue was manually minced and transferred into single cells suspensions using a gentleMACS Dissociator (Miltenyi) with DNase I (100U/mL, Applichem; Cat. No. A3778) and Collagenase IV (320u/mL, Worthington; Cat. No. LS004180). Cells were filtered through 100 μm and 70 μm nylon cell strainers (Greiner Bio-One; Cat. No. 89508-344 and 89508-340).
3
Intestinal Stem Cell Isolation and RNA-seq
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Embryonic small intestine or adult intestinal crypts (isolated with 5 mM EDTA) were dissociated with the TrypLE cell dissociation reagent (Thermo Fisher) and passed through a 40‐μm nylon cell strainer (Greiner). For sorting experiments involving the Lgr5Cre‐RosaTomato lines, tamoxifen was administered by i.p. 48 h before tissue harvesting. For RNAseq analysis, after cell gating and doublets exclusion, ISCs EGFP+ve cells were directly sorted using the FITC channel (Lgr5‐DTReGFP line) or were selected as recombined EGFP+ve/Tomato+ve cells using the FITC and PE channels (Lgr5Cre‐RosaTomato line). Sorted cells were collected over QIAzol lysis reagent (Qiagen).
4
Isolation and Sorting of Gastric Stem/Progenitor Cells
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Embryonic stomach samples or adult Lgr5-DTR antral glands, isolated as previously described (Barker et al., 2010 (link)), were dissociated with the StemPro Accutase cell dissociation reagent (Thermo Electron) and passed through a 40-μm nylon cell strainer (Greiner).
Fluorochrome-conjugated antibodies or relevant isotype controls were used for staining in PBS containing 2% BSA and 2 mM EDTA for 45 min on ice and sorted using a FACSAria I (BD Biosciences). For spheroid formation efficiency measurements, sorted cells were cultured ex vivo under ENR conditions. Quantification methods are detailed insupplementary Materials and Methods . For RNA-Seq analysis, Lgr5-DTR-EGFP+ cells were directly sorted using the FITC channel. Sorted cells were directly collected for RNA-Seq analysis over Qiazol lysis reagent (Qiagen). For adult Trop2+ and Lgr5-GFP+ sorting, cells were pooled (two pools of two HE-T samples, one pool of four HE-NT samples) to extract RNA from a total of 4000-8000 cells. For fetal Trop2+ cells, a mean of 30,000 cells were sorted in each independent experiment.
Fluorochrome-conjugated antibodies or relevant isotype controls were used for staining in PBS containing 2% BSA and 2 mM EDTA for 45 min on ice and sorted using a FACSAria I (BD Biosciences). For spheroid formation efficiency measurements, sorted cells were cultured ex vivo under ENR conditions. Quantification methods are detailed in
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