Spleens, lungs, and bronchoalveolar lavages (BAL) were collected after mice were sacrificed at indicated times with isoflurane/CO2. Lungs were isolated from the trachea intact to perform BAL and gain BAL fluids. BAL cells were separated through centrifugation and resuspended in PBS with 0.5% FBS and 0.1% NaN3. Lung parenchymal cells were isolated after digestion with 1mg/ml collagenase type II (Worthington, Columbus, OH, USA) and 0.1% DNase I (Sigma) in RPMI 1640 supplemented with 10% FBS for 30 min at 37°C. They were then passed through a 70-µm cell strainer (SPL Life science, Pocheon, Korea). Spleens were homogenized using frosted glass, and red blood cells were lysed using Red Blood Cell Lysing Buffer (Sigma). Homogenous cell suspensions were prepared by passing tissues through a 70-um cell strainer (SPL Life science) and resuspended in IMDM or RPMI supplemented with 10% FBS. For intravascular immunostaining, monoclonal anti-CD45-PerCP/Cyanine 5.5 (clone 30-F11) (BioLegend, San Diego, CA, USA) was prepared at a 2 µg/200 µl concentration and injected intravenously through the tail vein. Animals were euthanized and tissues were isolated 5 min after injection.
70 m cell strainer
Manufactured by SPL Life Sciences
Sourced in United States
The 70-µm cell strainer is a laboratory equipment used for filtering and separating cells. It features a mesh with 70-micrometer pore size designed to retain larger cells while allowing smaller particles to pass through.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Merck Group (1 mentions)
Collagenase type 2, by Worthington (1 mentions)
Red blood cell lysing buffer, by Merck Group (1 mentions)
70 um cell strainer, by SPL Life Sciences (1 mentions)
Accuri c6 plus flow cytometer, by BD (1 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
Rbc lysis buffer, by BioLegend (1 mentions)
Facsflow sheath fluid, by BD (1 mentions)
Fetal bovine serum (fbs), by Atlas Biologicals (1 mentions)
Dulbecco modified eagle medium (dmem), by Capricorn (1 mentions)
Collagenase type 1, by Merck Group (1 mentions)
Biocoll gradient solution, by Harvard Bioscience (1 mentions)
Collagenase type 1, by Thermo Fisher Scientific (1 mentions)
Minimum essential medium (mem), by Welgene (1 mentions)
Hep 2 cells, by American Type Culture Collection (1 mentions)
Red blood cell lysis solution, by Merck Group (1 mentions)
Rpmi 1640 medium, by Welgene (1 mentions)
Lsrii instrument, by BD (1 mentions)
Live dead fixable red dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Apc h7 conjugated anti cd4 antibody, by BD (1 mentions)
8 protocols using 70 m cell strainer
1
Multi-tissue Immune Cell Isolation
2
Splenocyte Isolation and Flow Cytometry
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Spleen cells were isolated at 14 days post infection (dpi). Homogenized single-cell suspensions were prepared in 6 mL of DMEM (Capricorn Scientific, Ebsdorfergrund, Hessen, Germany) supplemented with 10% FBS (Atlas Biologicals, Fort Collins, CO, USA) and 1% penicillin/streptomycin using gentleMACS™ Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were passed through a 70-µm cell strainer (SPL Life Sciences, Gyeonggi-do, South Korea), and centrifuged at 314× g for 7 min at 4 °C, followed by re-suspension and incubation in RBC lysis buffer (BioLegend, San Diego, CA, USA) at 4 °C for 5 min. After washing (314× g 7 min at 4 °C), cells were kept on ice in FACSFlow sheath fluid (BD Biosciences, San Jose, CA, USA) containing 0.05% FBS (Atlas Biologicals, USA) and Fc block (CD16/CD32 Fcγ III/II, BioLegend, San Diego, CA, USA) (1/1000 dilution) for 30 min in the dark at 4 °C. Next, 105 cells per sample were incubated for 30 min in the dark at 4 °C, with antibody cocktails specific for different splenocytes populations, followed by flow cytometry analysis using a BD Accuri™ C6 Plus flow cytometer (BD Biosciences, San Jose, CA, USA). The gating strategies used have previously been published [84 (link)], and the percentage of each population was determined by dividing the number of events within a specific gate, by the total number of events within live gate.
3
Isolation and Characterization of Immune Cells
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After the rats were euthanized, the skull was cut from anterior to posterior along the dorsal midline, and the brain was separated and fixed in 4% paraformaldehyde for immunohistochemical assay. The abdomen was dissected, and the liver and the kidneys were isolated. Blood samples were obtained from an abdominal artery puncture with heparin-coated 2 mL syringes and collected into tubes precoated with ethylenediaminetetraacetic acid (EDTA). The liver and the kidneys tissues were divided into two parts. One was fixed in 4% paraformaldehyde for immunohistochemical assay and the other was minced into 1 mm3 pieces on ice. After the minced tissues were washed, the tissues were digested with 1 mg/mL collagenase type 1 (Sigma-Aldrich, St. Louis, MO, USA) in 5 mL phosphate-buffered saline (PBS) at 37°C for 60 min. After incubation, the cells in the digested solution were filtered through a 70 µm cell strainer (SPL Life Science, Seoul, Korea). Lymphocytes were isolated from the blood, the liver and the kidneys, to assess ER stress and to detect ROS, using density-gradient centrifugation over a Biocoll gradient solution (Biochrom, Berlin, Germany).
4
Isolation of Primary Cell Cultures
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UL tissue pieces were treated with 1 mg/mL of collagenase type I (Invitrogen) for 1 h at 37 °C in a water bath and repeatedly suspended by gentle pipetting. The digested tissues were filtered through a 70-µm cell strainer (SPL Life Sciences, Seoul, Korea) and centrifuged at 3000 rpm. Cell pellets were replated and the medium was replaced with fresh medium every other day. The medium consisted of 1:1 mix of Dulbecco’s modified Eagle medium and Ham’s F-12 medium (DMEM/F12) without phenol red, 10% fetal bovine serum (FBS), 1% insulin–transferrin–selenium (ITS), and 50 U/mL penicillin–streptomycin. All these reagents were purchased from Invitrogen.
5
Lung Viral Titer Quantification
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On day 5 after the RSV challenge, all mice were sacrificed by CO2 euthanasia and single-cell suspensions of lung tissue were obtained using MEM (Welgene) and a 70 µm cell strainer (SPL). The cells and supernatants were separated by centrifugation at 1,600 rpm and 4℃ and the supernatants were incubated at 37℃ for 5 days with HEp-2 cells (ATCC). Viral titers were recorded as PFU/g of lung tissue, with a limit of detection of 100 PFU/g.
6
Bone Marrow Cell Isolation and Transplantation
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Eight-week-old C57BL/6 male mice were sacrificed by cervical dislocation and the lower limbs (femurs and tibias) were collected. BMCs were harvested, following dissection and cleaning of the bones, by removing the ends of each bone and flushing the medullary cavities with serum-free RPMI-1640 medium (Welgene, Inc.) using a 25-gauge needle (Korea Vaccine, Ansan-si, Korea). A single cell suspension was produced by passing the BM suspension through a sterile 70-µm cell strainer (SPL Life Sciences, Pocheon, South Korea) and the filtrate was centrifuged at 300 × g for 5 min. Isolated BMCs were incubated in red blood cell (RBC) lysis solution (Sigma-Aldrich; 0.15 M NH4Cl, 10 mM NaHCO3 and 10 mM disodium EDTA, all from Sigma-Aldrich) for 2 min followed by two washes with PBS. Following resuspension with PBS, 2×106 cells/200 µl were intravenously injected into recipient mice using 1-ml syringes (Sungshim, Bucheon-si, Korea).
7
Isolation and Analysis of Nasal Lymphocytes
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Nasal tissues, including NPs and ethmoidal mucosa, were gathered and minced into small pieces to isolate lymphocytes. The minced tissues were mechanically homogenized. The homogenates were filtered through a 70-µm cell strainer (SPL Lifesciences, Pocheon, Korea). The filtered cells, including the nasal mucosal lymphocytes, were collected and cryopreserved until the time of analysis. The cryopreserved cells were thawed for analysis. At first, to exclude dead cells from analysis, cells were treated with the LIVE/DEAD Fixable Red Dead-Cell Stain Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. After washing, Cells were stained with BD horizon V500-conjugated anti-CD3 antibody (BD Biosciences, San Jose, CA, USA), APC-H7-conjugated anti-CD4 antibody (BD Biosciences), APC-conjugated anti-CCR4 antibody (R&D Systems, Minneapolis, MN, USA), and PE-conjugated anti-CXCR3 antibody (R&D Systems) in the dark for 30 min at 4°C to stain chemokine receptors on CD4+ T cells. Cells were washed and analyzed by multicolor flow cytometry. Flow cytometry was performed with an LSR II instrument (BD Biosciences), and data were analyzed with FlowJo software (Treestar, Ashland, OR, USA).
8
Isolation and Characterization of Intestinal Intraepithelial Lymphocytes
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Isolation of IEL was performed as previously described with slight modification (15 ). Briefly segments of the duodenum (about 7 cm) were opened longitudinally and cut into roughly 2-cm fragments. To remove the intestinal mucus, the cut tissue fragments were initially washed with fresh RPMI followed by pre-warmed PBS/EDTA/DL-Dithiothreitol (DTT) (Sigma-Aldrich, USA) solution and then incubated in a shaking water bath at 37°C for 30 min. The samples were later homogenized using a 70-µM cell strainer (SPL Life Science, Korea). Dead cells from the single cell suspension were discarded following Histopaque-1119 (Sigma Aldrich, USA) density gradient centrifugation and the cell viability was determined using Trypan blue dye exclusion method and Scepter 2.0. Finally, the cells were divided into two parts, one part was stained for immunophenotyping of IEL and the second part was processed by magnetic activated cell sorting (MACS) (Miltenyi Biotec, Germany) for selective characterization.
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