Cells from mouse spleen and lymph nodes were isolated by mashing organs through a 40-μm strainer (Corning) in RPMI supplemented with 10 % heat-inactivated fetal bovine serum (FBS). Red blood cells were lysed using an ammonium chloride solution (Stemcell Technologies). Cells were washed and resuspended in FACS buffer (PBS, 2 % FBS and 1 mM EDTA), followed by staining for 30 min at 4 °C. The stained cells were then washed and analyzed using a BD LSR Fortessa, followed by data analysis using FlowJo v10 software (Tree Star). Fluorescein-labeled anti-CD4 scFv (GK 1.5), prepared as described below, and the following fluorescent dye-conjugated antibodies were used for staining: anti-CD3 (145-2C11) anti-CD4 (RM4-5), anti-CD8α (53-6.7), anti-CD19 (eBio1D3) and anti-CD45.1 (A20). Cell viability was measured by an Aqua dead cell stain kit (ThermoFisher Scientific).
40 μm strainer
Manufactured by Corning
Sourced in United States
The 40-μm strainer is a laboratory equipment designed for filtration and separation purposes. It features a mesh with a pore size of 40 micrometers, allowing the passage of smaller particles while retaining larger ones. The strainer is made of durable materials suitable for use in various laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Tissue plus o c t compound, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Meiji Seika Pharma (2 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Horse serum, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Cd45 microbeads, by Miltenyi Biotec (2 mentions)
Aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Ammonium chloride solution, by STEMCELL (1 mentions)
Collagenase type 1, by Thermo Fisher Scientific (1 mentions)
100 μm cell strainer, by Corning (1 mentions)
Novaseq 6000 system, by Illumina (1 mentions)
Facsaria 2, by BD (1 mentions)
Dulbecco s modified eagle s medium dmem f12, by Thermo Fisher Scientific (1 mentions)
Collagenase 1, by Thermo Fisher Scientific (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Cryostat, by Leica (1 mentions)
Accuri flow cytometer, by BD (1 mentions)
13 protocols using 40 μm strainer
1
Isolation and Phenotyping of Murine Immune Cells
2
Isolation and Differentiation of Primary Murine Adipocytes
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BAT, iWAT, and gWAT from 8 weeks old male WT and TMEM86A AKO mice were used for primary cell studies. Adipose tissues were isolated, washed, minced, and digested with collagenase type I (Gibco, 2 mg/mL, cat#17100-017) in KRBB buffer containing 3% BSA at 37 °C. Fully dissociated preparations were then passed through a 100 μm cell strainer (Corning) and centrifuged at 300 × g for 5 min. Floating adipocytes were collected for further analysis. Red blood cells were lysed with RBC lysis buffer (15.5 mM NH4Cl, 1.2 mM NaHCO3 and 50 mM EDTA, pH 7.4) and pellets were then washed with DMEM containing 20% FBS and 1% P/S for two times. Stromal vascular fraction (SVF) was filtered through 40 μm strainer (Corning) and distributed onto the plate-format of interest. The isolated adipocyte precursors were washed with PBS and the media was replaced each day. When the adipocyte precursors were 100% confluent, cells were induced to differentiation medium containing IBMX (0.5 mM), dexamethasone (1 μM), insulin (10 μg/mL), and indomethacin (0.2 mM) for 3 days and then induced to maintenance medium containing insulin (10 μg/mL) for 3 days. Fully differentiated primary adipocytes were further analyzed with immunoblot and oxygen consumption measurements.
3
Single-cell RNA sequencing of diverse cell types
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Fresh isolated cells from bone marrow, placenta, and fat were filtered with 40 μm strainer (Corning) and resuspended by 0.04% BSA in HBSS (Thermo Fisher Scientific) at the concentration of 1 × 106 cells/mL. Single-cell GEM (Gel Beads-in-Emulsion) was produced with 10 × Genomics Chromium platform according to the instructions. Finally, the libraries were sequenced with Illumina NovaSeq 6000 System (paired-end mode). Raw data processing, mapping, filtering, and UMI count matrix generation were conducted with 10xGenomics pipeline Cell Ranger (v2.1.0). Then, the gene-cell barcode matrix was further analyzed with Seurat package in R (v 4.0.0).
4
Isolation and FACS of CX3CR1+ Cells
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Freshly isolated pallial walls derived from E14 male and female CX3CR1-GFP mice were treated with trypsin (0.05%, 3 min at 37°C). Dissociated pallial cells were filtered through a 40 μm strainer (Corning) to eliminate all remaining cell clumps, and then resuspended in DMEM containing 5% fetal bovine serum (Invitrogen), 5% horse serum (Invitrogen), and penicillin/streptomycin (50 U/ml, each; Meiji Seika Pharma). CX3CR1-GFP+ cells were sorted through a 100-μm nozzle by FACS Aria II (BD Biosciences). The drop delay was optimized using BD Biosciences Accudrop beads (BD Biosciences).
5
Isolation and Culture of Adipose-Derived Stem Cells
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GFP male mice (8–10 weeks old) were sacrificed and disinfected with 75% ethanol. Adipose tissue in bilateral groins was isolated, and vessels were removed. Adipose tissue was digested with 0.1% collagenase I (Invitrogen, Carlsbad, CA, USA) for 40 min at 37 °C with gentle agitation and stopped using ADSC complete medium [Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco, Gaithersburg, MD, USA) supplemented with 10% foetal bovine serum (FBS; Gibco), 1× GlutaMAX (Gibco), and 1× penicillin-streptomycin (Gibco)]. Cells were collected by centrifugation at 1100 rpm for 10 min and resuspended using ADSC complete medium. After filtering the cell resuspension with a 40-μm strainer (Corning, Corning, NY, USA), cells were seeded in T25 flasks and cultured at 37 °C in an incubator with 5% CO2. ADSCs at low passage (< 8) were used in this study.
6
Tissue Harvesting and Processing for Flow Cytometry and Immunohistochemistry
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Mice were euthanized with CO2 and then transcardially perfused with 20 mL 1 x PBS. Spleens for flow cytometry were harvested, placed into cold cRPMI, then mechanically homogenized and washed through a 40 μm strainer (Corning). Cells were resuspended in RBC lysis solution (0.16 M NH4Cl) for 2 min. Cells were then washed, resuspended, and kept on ice until acquisition. Brains for immunohistochemistry were removed and kept in 4% PFA for 24 hr and then cryoprotected with 30% sucrose for 3 days. A 4 mm coronal section of brain tissue that surrounded the site of injury was removed using a brain sectioning device and then frozen in Tissue-Plus OCT compound (Thermo Fisher Scientific). Fixed and frozen brains were sliced (50 μm thick sections) with a cryostat (Leica) and kept in PBS + 0.05% sodium azide in PBS at 4 °C until further use.
7
Isolation and Purification of Pallial Cells
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Freshly isolated pallial walls were treated with trypsin (0.05%, 3 min at 37 °C). Dissociated pallial cells were filtered through a 40-μm strainer (Corning, Corning, NY, USA) to eliminate all remaining cell debris and then resuspended in DMEM containing 5% fetal bovine serum (FBS) (Invitrogen, Waltham, MA, USA), 5% horse serum (HS) (Invitrogen), and penicillin/streptomycin (50 U ml−1, each) (Meiji Seika Pharma Co., Ltd., Tokyo, Japan). CX3CR1-GFP+ cells or Gadd45g-d4Venus+ cells were sorted through a 100 μm nozzle using FACSDiva software version 8.0 on FACS SORP Aria II (BD Biosciences, Franklin Lakes, NJ, USA). The drop delay was optimized using BD Biosciences Accudrop™ beads (Cat#345249, BD Biosciences) according to the manufacturer’s recommendations. Cerebral wall cells were gated on a forward scatter (FSC)/side scatter (SSC) plot (Fig. 6b ; Supplementary Figs. 3a and 14 ). Debris and dead cells were excluded, and then CX3CR1-GFP+ or Gadd45g-d4Venus+ cells were further gated for sorting. For RNA-Seq and FACS analyses, for which we needed to prepare many microglia, CD11b+ cells were collected using a magnetic bead separation (MACS) system (Cat#130–093–634, Miltenyi Biotec, Bergisch Gladbach, Germany) from ICR mouse cerebral wall cells.
8
Isolation and Analysis of Colonic Lamina Propria Leukocytes
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Leukocytes from proximal colonic lamina propria were isolated after washes with 2 mM EDTA and digestion with collagenase V from Clostridium histolyticum (1 mg/ml; Sigma-Aldrich) (26 (link)). The cells were washed through 40-μm strainers (Corning, Corning, NY, USA) and stained with CD4 (FITC) and CD25 (APC) (1:100; BD Biosciences). Positive populations were determined by labeling with single antibodies. A minimum of 10,000 events per sample were acquired on a BD Accuri Flow Cytometer. The results were expressed as percentages of positive cells normalized by controls from each experiment.
9
Colonic Cell Isolation and Stimulation
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After euthanasia by overexposure to isoflurane, colons from C57BL6 mice were opened longitudinally and washed with supplemented phosphate-buffered saline (10,000 μg/ml penicillin/streptomycin and 50 μg/ml gentamycin). Under a sterile hood, the tissues were fragmented, washed in Hank’s salt solution buffer without calcium/magnesium for 20 min (twice), and digested with collagenases from C. histolyticum (types II and IV, 0.5 mg/ml; Gibco, Waltham, MA, USA). The digested tissue was washed twice through 40-μm strainers (Corning) and the pellets were counted and resuspended at the Roswell Park Memorial Institute (RPMI + 1% FBS). Cells were seeded in a 96-well plate (2 × 105/well) and treated with 200 ng/ml lipopolysaccharide (LPS; Sigma-Aldrich) 30 min before IFX (0.1, 1.0, or 10.0 μg/ml). Controls were untreated or treated with those IFX doses. After 24 h, the supernatants were collected to dose secreted AnxA1 (MyBioSource) according to the manufacturer’s instructions.
10
Murine Tumor Immune Cell Profiling
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Murine tumors were pushed through 40μm strainers (Corning). CD45+ cells were magnetically enriched using CD45 Microbeads (Miltenyi Biotech). Enriched cells were stained as described in Supplementary Figure 7 and sorted in culture medium or in RLT buffer for RNA extraction (RNeasy micro kit, and DNase set Qiagen). Spleen cell was depleted for B and T cells using Dynabeads (Invitrogen), anti-B220 (553084, BD Biosciences) and anti-CD3 (100202, Biolegend), then stained and sorted as above. MMLV (Thermo) was used for reverse transcription and SYBR Green (Thermo) for qPCR (CFX96 Touch™ Real-Time PCR Detection System, Bio-Rad). Alternatively, one step RT-PCR was performed from purified RNA using QuantiTect SYBR Green (Qiagen). RT-PCRs where performed with primers sets purchased from Sigma-Aldrich (M_CCL2_1, M_CCL4_1, M_CCL7_1, M_CXCL3_1, M_CXCL9_1, M_CEBPB_1, M_NCOR2_1) and mouse B2M, Fw: 5’-TTCTGGTGCTTGTCTCACTGA-3’ and Rv: 5’-CAGTATGTTCGGCTTCCCATTC-3’.
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