To maximize the SVF yield from both sample sources and properly prepare cells for flow cytometry, we equally treated LA and MLA samples with 1% collagenase type I in Dulbecco’s Modified Eagle Medium (D-MEM) (both from Sigma-Aldrich, Saint Louis, MO, USA) in a shaking bath at 37 °C for 45 min. After 1:2 dilution with 2% fetal bovine serum (Biosera, Nuaille, France) in D-MEM (Sigma-Aldrich), samples were filtrated through a 100 µm-cell strainer (BD Falcon, Corning, NY, USA) and centrifuged at 300 g for 10 min at RT. Supernatants were discarded and cell pellet resuspended in 1 mL of the VersaLyse solution (Beckman Coulter, Miami, FL, USA). After 10 min, samples were filtered through a 40 µm-cell strainer (BD Falcon, Corning, NY, USA), centrifuged at 300 g for 10 min at RT and the cell pellet resuspended in D-MEM (Sigma-Aldrich). The cells were counted on the Sysmex XT1800 counter (Sysmex, Kobe, Japan).
100 m cell strainer
Manufactured by BD
Sourced in United States, Germany
The 100 μm cell strainer is a laboratory equipment designed for filtering and separating cells. It features a mesh screen with a pore size of 100 micrometers, allowing the passage of single cells and smaller cellular components while retaining larger particles and cell aggregates.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Dnase 1, by Roche (4 mentions)
Dnase 1, by Merck Group (4 mentions)
40 m cell strainer, by BD (3 mentions)
Facsdiva, by BD (3 mentions)
Percoll, by GE Healthcare (3 mentions)
Collagenase d, by Roche (3 mentions)
Collagenase type 4, by Thermo Fisher Scientific (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Heparin, by Merck Group (3 mentions)
Insulin, by Merck Group (3 mentions)
Hydrocortisone, by Merck Group (3 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (3 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (3 mentions)
Fc block, by BD (3 mentions)
Liver perfusion medium, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
Dnase, by Merck Group (2 mentions)
91 protocols using 100 m cell strainer
1
Optimizing SVF Yield for Flow Cytometry
2
Primary Keratinocyte Isolation from Mouse Tail
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Primary keratinocytes were isolated from the skin of adult mice tail. Briefly, the skin from the tail was collected and incubated at 4°C for 16 h in a solution of 5 U/mL Dispase (STEMCELL) supplemented with 1% PNS (Gibco) and 0.5% Gentamicin (Gibco). The epidermis was separated from the dermis and treated with 0.2% trypsin (Gibco) for 5 min at 37°C. The reaction was stopped with FCS, and cells were collected by crushing the epidermis with a 100-µm cell strainer (Falcon). 7.5 × 105 cells/mL were plated in a 6-well plate coated with Type I collagen (STEMCELL). Cells were grown for 8 days at 37°C in CnT-57.S medium (CELLnTEC) supplemented with 1% PNS and 0.5% Gentamicin. Medium were removed and cells detached with 200 µL 0.2% trypsin for 5 min at 37°C. Cells were counted and plated at 0.3 × 106 cells/mL in a 96-well plate coated with Type I collagen.
3
Isolation of Canine Adipose-Derived Stem Cells
4
Comprehensive Immune Cell Profiling
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Splenocytes or lymph node cells were extracted into single cell suspension in 1× PBS, and RBCs lysed by incubation in ACK lysis buffer (Biofluids), single cell suspensions were passed through 100 µm cell strainer (BD Falcon). Cells were washed and FcγRII/III blocked with αCD16/32 (eBiosciences). Surface markers were stained with fluorochrome‐conjugated mAbs toCD3, CD4, CD69, CCR7, IL7Rα, CD62L, CD44, CD45, CD103, CD25, CD8α, CD11b, CD11c, CD19, CD62L, CD80, CD86, CD117 (c‐kit), DX5, F4/80, MHC Class II (I‐A/I‐E), (eBiosciences, BD Pharmingen, Biolegend, AbD Serotec). Treg cells were further stained by intracellular staining of Foxp3 with a kit according to manufacturer's instructions (eBioisciences). Samples were acquired on the LSR II quad‐laser cytometer running FACSDiva (BD Immunocytometry).
5
Establishment of KPCML1 Liver Metastasis Cell Line
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A visible KPC liver metastatic nodule was excised in a sterilized animal manipulation hood and placed in ice cold DMEM + 10% FBS. The nodule was transferred in media into a tissue culture hood. After rinsing the tissue with PBS to remove excess blood, the nodule was minced into approximately 3.0 mm pieces and digested in an enzyme solution (0.05 mg/ml collagenase I, 0.05 mg/ml collagenase IV, Hyaluronidase 0.025 mg/ml, DNase I 0.01 mg/ml in HBSS) for 30 min at 37°C in a tissue culture incubator. The supernatant was transferred to a 50 ml conical tube containing full DMEM media and the remaining pieces were further digested for another 15 min. All digested supernatants were pooled together and passed through a 100 µM cell strainer (BD FALCON). After washing the strainer twice with DMEM + 10% FBS, cells were cultured in a 10 cm tissue culture dish until they reached 70 to 80% confluence. Cells were then harvested by trypsinization and split at 1:80 for subculturing. After 3 rounds of similar subculturing and when cells were observed to grow uniformly on tissue culture dishes, cells were expanded into multiple dishes and subsequently cryopreserved in DMEM + 10% FBS + 10% DMSO solution. The generation of this new cell line was referred to as KPCML1 (KPC M etastasis L arge nodule 1 ).
6
Evaluating Fistula-Derived Immune Cells in CD
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CD fistula samples were obtained from fistulizing CD patients undergoing surgery (seton placement/removal, or inspection) at the Amsterdam UMC, location AMC. Fistula scrapings were mechanically digested by mashing and flushing through a 100 µm cell strainer (BD Falcon, Franklin Lakes, NJ, USA) placed on a 50 mL tube (Sarstedt, Germany), and immune cells were isolated using Ficoll isolation [26 ]. Immune cells were incubated for 16 h with a concentration range of 0.0025, 0.01, 0.04, 0.625, 2.5, and 10 µM of either GSK3361191 (ESM-iBET), non-hydrolysable control GSK3235220 (iBET), or DMSO resolved in RPMI medium (Thermofisher Scientific, Waltham, MA, USA). After incubation, the cells were either collected for cytokine analysis by CBA or flow cytometric analysis of intracellular TNFα. Cytokine data is visualized by normalizing the actual measured values to the DMSO control to correct for the biological variation in every individual patient.
7
Isolation of Non-Parenchymal Cells from Mouse Livers
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Non-parenchymal cells were isolated from mouse livers at 40 h post-infection. Cells were obtained by adapting a method previously described (21 (link)). Briefly, liver lobes were removed and perfused with liver perfusion medium (Gibco; Invitrogen) with 750 mg/l of Collagenase H (Roche) at 37°C. The resulting suspension was filtered through a 100 µm cell strainer (BD Falcon). The dissociated cells were suspended in liver perfusion medium and centrifuged for 10 min at 1,500 rpm, resuspended in RPMI complete medium (Gibco; Invitrogen), mixed in Percoll (GE Healthcare) solution to give a final concentration of 30% Percoll, and then centrifuged at 2,000 rpm for 10 min. The cell pellet was resuspended in RPMI and carefully laid on 30% Percoll solution and centrifuged at 2,000 rpm for 10 min. The cell pellet collected was washed and resuspended in ACK (NH4Cl 0.15 M, KHCO3 10 mM, Na2EDTA⋅2H2O 0.1 mM and pH 7.2) for 3 min to lyse remaining erythrocytes. Cells were washed and centrifuged at 800 rpm for 20 s to discard the remaining hepatocytes; the supernatant was recovered, and NPCs were collected at 1,500 rpm for 5 min.
8
Flow Cytometry Analysis of Tumor-Infiltrating Immune Cells
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Macrophages in cultures were incubated with fluorescence-labeled antibodies against CD43, CD86, and CD206 (BioLegend, San Diego, CA, USA) and colony-stimulating factor 2 receptor alpha (CSF2RA; Santa Cruz Biotechnology, Dallas, TX, USA) for 20 min in the dark. For analysis of immune cell populations in tissues, freshly excised tumors were fragmented into several pieces. The fragmented tissues were further minced into 2–3 mm3 pieces and incubated with collagenase D (Roche, Basel, Switzerland) and DNase (Merck), at 37°C for 40 min. The tissue samples were filtered through a 100 µm-cell strainer (Falcon, Corning, NY, USA) to collect the digested cells. Dead cells and cellular debris were removed using Ficoll® (GE Healthcare, Buckinghamshire, UK) gradient centrifugation. Collected cells were re-suspended in phosphate-buffered saline containing FBS and incubated with fluorescence dye-labeled antibodies against CD3, CD4, CD8, CD11b, CD45, CD86, CD206, F4/80, and Gr1 (BioLegend) for 30 min in the dark. Primary antibodies were diluted at a ratio of 1:100. After gating for CD45+ cells, the cells were analyzed for the corresponding markers. At least 10,000 events were analyzed using the FACSCalibur™ cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Data were evaluated using CellQuest™ Pro (BD Biosciences).
9
Isolation and Culture of Murine Immune Cells
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For bone marrow-derived macrophages and dendritic cells (DCs), bone marrow was obtained from mouse femurs and grown for 6 to 8 days in RPMI 1640 in the presence of 10% L929 conditioned medium [32] (link) and collected 7 days later by scraping. Bone marrow-derived DCs were cultured in the presence of GM-CSF (20 ng/ml) and collected 6–8 days after culture [33] (link). For the splenic T-cells and B-cells, spleens were harvested, mechanically homogenized, and filtered through a 100 µm cell strainer (Falcon). Erythrocytes were lysed with cold ACK lysing buffer (Cellgro) for 5 min, and the cell suspension was washed with complete medium. T cells were purified with negative selection enrichment columns (R&D Systems) following the manufacturer’s recommendations. B cells were enriched by negative selection using magnetic isolation kits, as per manufacturer’s instructions (Miltenyi Biotec). This process resulted in cell purities of >97% as determined by flow cytometry.
10
Isolation and Characterization of Mast Cells
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Meningeal tissue was carefully harvested from the skullcaps as previously described25 (link) using an 8 mm biopsy punch (Integra) and extracted by gently scraping the tissue from the bone. Tissue was placed in cold RPMI media (Cytiva) supplemented with 10% FBS (Gibco) and 1% PenStrep (Gibco) followed by incubation at 37 °C while rotating at 12 rpm for 30 min in an Incubated Rotator (Benchmark). The tissue was suspended in a digestion mix consisting of Dispase II at 1.25 mg/mL (Sigma), Collagenase II at 2 mg/mL (Gibco), and Collagenase IV at 2 mg/mL (Gibco). A 100 µM cell strainer (Falcon) was used to filter cells from the digested tissue. Dead cells were stained using Live/Dead Fixable Aqua Dead Cell Stain Kit (Invitrogen).
Cells were treated with Fc block (BioLegend) for 10 min before the addition of all the following antibodies to each sample: Brilliant Violet 605 anti-c-kit (BioLegend), APC-Cy7 anti-CD45 (BioLegend), FITC anti-FcεRIα (BioLegend) for the MrgprB2+ mice. For the X2+ mice, PE anti-X2 (BioLegend) was added. Data were collected using a FACSCelesta Flow Cytometer (BD) and analyzed using FlowJo (TreeStar). Mast cells were gated as live CD45+ c-Kit+ FcεRI+ and tdT+ (MrgprB2+ mice) or MRGPRX2+ (X2+ mice).
Cells were treated with Fc block (BioLegend) for 10 min before the addition of all the following antibodies to each sample: Brilliant Violet 605 anti-c-kit (BioLegend), APC-Cy7 anti-CD45 (BioLegend), FITC anti-FcεRIα (BioLegend) for the MrgprB2+ mice. For the X2+ mice, PE anti-X2 (BioLegend) was added. Data were collected using a FACSCelesta Flow Cytometer (BD) and analyzed using FlowJo (TreeStar). Mast cells were gated as live CD45+ c-Kit+ FcεRI+ and tdT+ (MrgprB2+ mice) or MRGPRX2+ (X2+ mice).
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