Cerebral microvessels were isolated from the ischemic hemispheres of mice that had undergone tMCAO surgery. Briefly, the hemispheric brain tissue was minced and homogenized in disaggregation buffer (15 mM HEPES, 150 mM NaCl, 4 mM KCl, 3 mM CaCl2, 12 mM MgCl2, 0.5% BSA and protease inhibitors). The homogenate was centrifuged at 3200 rpm for 10 minutes. The pellet was resuspended in 20% dextran and centrifuged for 30 minutes at 3000 rpm and 4ºC. The pellet was resuspended in PBS and filtered through a 100-μm nylon cell strainer (Corning® cell strainer, USA), followed by a new filtration through a 20-μm nylon mesh (Corning® cell strainer, USA). Retained (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. microvessels on the mesh were washed with RIPA buffer (Abcam, UK) and stored until further analysis. Protein concentration was determined using BCA protein assay kit (Thermo Scientific, USA) and 40 μg of lysate were loaded in 15% acrylamide gels. Antiglial fibrillary acidic protein (GFAP) (1:1000; Abcam, UK) was used as a control antibody for validation of succeeded vessel enrichment (Supplemental fig. 1).
Cell strainer
Manufactured by Corning
Sourced in United States, United Kingdom
The cell strainer is a laboratory equipment used for the filtration and separation of cells from a cell suspension. It is a mesh-based device that allows the passage of single cells while retaining larger cell clumps, tissue fragments, or cell aggregates.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (9 mentions)
Trypsin edta, by Thermo Fisher Scientific (7 mentions)
Dmem f12, by Thermo Fisher Scientific (7 mentions)
Dnase 1, by Merck Group (6 mentions)
Ack lysing buffer, by Thermo Fisher Scientific (6 mentions)
Dnase 1, by Roche (5 mentions)
Matrigel, by Corning (5 mentions)
Tryple express, by Thermo Fisher Scientific (5 mentions)
Streptomycin, by Thermo Fisher Scientific (5 mentions)
Rpmi 1640, by Thermo Fisher Scientific (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Collagenase type 1, by Merck Group (4 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Collagenase 2, by Merck Group (3 mentions)
Rnase inhibitor, by Takara Bio (3 mentions)
Papain, by Worthington (3 mentions)
10 ml syringe, by BD (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Collagenase d, by Roche (3 mentions)
160 protocols using cell strainer
1
Isolation of Cerebral Microvessels from Ischemic Mice
2
Quantifying Bacterial Encapsulation in Alginate
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Alginate and alginate-MA
beads were separated from suspension via
filtration (Cell strainer, 40 μm mesh, Corning) and washed with
excess 20 mM CaCl2 solution at least 4 times to remove
nonencapsulated bacteria. The washing solution was collected and poured
on agar plates. The agar plates were incubated at 37 °C for 24
h. The number of bacteria in the washing solution was evaluated by
counting the colonies on LB agar plates. The alginate and alginate-MA
beads were resuspended in fresh LB medium and stored at 37 and 4 °C,
respectively. The bead concentration was about 9000 beads/mL. For
storage at 37 °C, the suspension was separated from beads via
filtration (Cell strainer, 40 μm mesh, Corning) after 2, 4,
and 6 h incubation. The bead-free suspension was serially diluted
in Ringer’s solution, and the number of bacteria was evaluated
by counting the colonies on LB agar plates after incubation at 37
°C for 24 h. For storage at 4 °C, the number of bacteria
in the suspension was evaluated as described above after 1, 3, 14,
and 35 days incubation.
beads were separated from suspension via
filtration (Cell strainer, 40 μm mesh, Corning) and washed with
excess 20 mM CaCl2 solution at least 4 times to remove
nonencapsulated bacteria. The washing solution was collected and poured
on agar plates. The agar plates were incubated at 37 °C for 24
h. The number of bacteria in the washing solution was evaluated by
counting the colonies on LB agar plates. The alginate and alginate-MA
beads were resuspended in fresh LB medium and stored at 37 and 4 °C,
respectively. The bead concentration was about 9000 beads/mL. For
storage at 37 °C, the suspension was separated from beads via
filtration (Cell strainer, 40 μm mesh, Corning) after 2, 4,
and 6 h incubation. The bead-free suspension was serially diluted
in Ringer’s solution, and the number of bacteria was evaluated
by counting the colonies on LB agar plates after incubation at 37
°C for 24 h. For storage at 4 °C, the number of bacteria
in the suspension was evaluated as described above after 1, 3, 14,
and 35 days incubation.
3
Spleen Cell Proliferation and Cytokine Assays
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Spleens from mice adoptively transferred with DCs were aseptically dissected after euthanasia and spleen cells were separated using a 40 μm cell strainer (Corning Cell Strainer, Corning Incorporated, Durham, NC, United States). A cell suspension was prepared and transferred to 96-well plates at 105 cells/well followed by stimulation with concanavalin A (Con A-0.5 μg/mL final concentration; Sigma-Aldrich, St Louis, MO, United States) or heat-killed R. rickettsii (HKRr−2 × 105 bacteria/well) and incubated at 37°C under 5% CO2. After 48 h, 25 μL of 0.01% resazurin (prepared in complete medium) were added to culture cells and after additional 24 h, the culture absorbance at 570 and 600 nm was determined and used to indirectly evaluate cell proliferation as previously described (31 (link), 36 (link), 49 (link)).
In another set of experiments, spleen cells were distributed into 24-well plates at 2.5 × 106 cells/well followed by stimulation with 5 × 106 cells of HKRr/well. After 72 h of incubation at 37°C under 5% CO2, the concentration of IFN-γ and IL-4 was evaluated in cell-free supernatants according to manufacturer's instructions (BD OptEIA™ ELISA Set-BD Biosciences).
In another set of experiments, spleen cells were distributed into 24-well plates at 2.5 × 106 cells/well followed by stimulation with 5 × 106 cells of HKRr/well. After 72 h of incubation at 37°C under 5% CO2, the concentration of IFN-γ and IL-4 was evaluated in cell-free supernatants according to manufacturer's instructions (BD OptEIA™ ELISA Set-BD Biosciences).
4
Migration and Invasion Assays of Cells
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Cells were dissociated with trypsin (Gibco) to form a single cell suspension using a 40-μm Corning® Cell Strainer (Corning, Inc., Corning, NY, USA) and counted with a TC20™ Automated Cell Counter (Bio-Rad Laboratories). Migration assays were performed using Falcon® Cell Culture Inserts and Companion Plates in a 24-well plate with an 8.0-µm Transparent PET Membrane (Falcon, Corning, Inc.). Cells (5 × 105) were suspended in 500 μL of serum-free medium and seeded into the upper chamber with or without 10 µM Y-27632 (Selleck Chemicals). The lower chamber was filled with a medium containing 20% fetal bovine serum. After incubation for 24 h at 37°C, in an atmosphere containing 5% CO2, migrated cells were fixed in 100% methanol for 10 min and stained with Giemsa Stain Solution (FUJIFILM Wako) for 30 min. The migrated cells were counted in five random fields per sample under a microscope at 200× magnification (Olympus Corporation, Tokyo, Japan). Invasion assays were performed using the same method as that used for the migration assays, with the following exceptions: 1 × 105 cells were seeded into the upper chamber of a Matrigel-coated transwell chamber (Corning® BioCoat™ Matrigel® Invasion Chambers 24 Well, Corning, Inc.), and invaded cells were counted after incubation for 48 h.
5
Splenocyte Isolation from Murine Tissue
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To obtain single-cell suspensions, spleens were passed through a 70-mm por size filter (Corning cell strainer; Corning, Sigma Aldrich) and centrifuged over a Ficoll gradient (Histopaque®-1083) in 15 mL falcon tubes. The different components were separated by centrifugation (400 g 30 min, RT, without brake). The upper fraction was removed and the cells from the interface were collected with a pasteur pipette. The cells were diluted with RPMI to 5 ml for washing (400 g, 10 min, RT), counted and re-suspended in the R10 culture medium to achieve a concentration of 5*106 cell/well.
6
Characterization of Transgenic NOD Mice
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Transgenic NOD.B6-Tg(HLA-A2.1)1Enge/DvsJ mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and maintained in the specific-pathogen–free facilities at the Molecular Medicine and Chronic Diseases Research Centre (Santiago de Compostela). Glucosuria was determined weekly from 8 wk of age onward using Medi-Test Glucose Strips (Macherey-Nagel, Düren, Germany). Diabetes was diagnosed when glucose levels were ≥500 mg/dl (27.8 mM) in 2 consecutive measures. All experimental procedures were approved by the corresponding ethics committee. Splenocytes were stained with the BB7.2 antibody to confirm A2.1 expression.
To obtain single-cell suspensions, spleens were passed through a 70-μm pore size filter (Corning cell strainer; Corning, Corning, NY, USA) and centrifuged over a Ficoll gradient.
To obtain single-cell suspensions, spleens were passed through a 70-μm pore size filter (Corning cell strainer; Corning, Corning, NY, USA) and centrifuged over a Ficoll gradient.
7
Isolation of Decidua Cell Suspensions
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Decidua cell suspensions were prepared from decidua tissues by a modification of Petrovic's method [21 (link)]. After the fragments of decidua were washed with PBS and the maternal blood was removed, the tissues were identified macroscopically and minced with scissors and then incubated at 37°C in 5% CO2 with continuous agitation in RPMI-1640 medium (GIBCO, GrandIsland, NY, USA) containing 0.25% Trypsin-EDTA (GIBCO). After 90 minutes, the cell suspension was filtered through a 100 μm nylon mesh Corning Cell Strainer (CORNING, Tewksbury, MA, USA), washed three times with RPMI-1640, and centrifuged at 400 g. The cell pellets were resuspended in RPMI-1640 containing 10% fetal bovine serum (FBS) (SIGMA, St Louis, MO, USA) and then layered onto Lymphoprep (AXIS-SHILD, Oslo, Norway) solution and centrifuged for 20 minutes at 400 g. Cells aspirated from the interface were washed three times with PBS and adjusted to a concentration of 1X107 cells/ml in RPMI. Cell viability was confirmed to be >95% by trypan blue dying. Peripheral blood lymphocytes were also separated using Lymphoprep.
8
In Vivo Genotoxicity Evaluation in Liver
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In vivo rGOM genotoxicity was examined in liver tissue of Wistar rats used for the in vivo biocompatibility assay. Briefly, hepatic tissue from animals euthanized at 21 days was washed 3 times with cold PBS. The tissue was then carefully chopped and incubated with trypsin/EDTA 0.25 % at 37°C for 30 min. After this time the sample was filtered through a Corning cell strainer® (Corning, New York, New York, USA) and the suspension was centrifuged at 400 rcf for 6 min. The obtained pellet was washed 3 times with PBS and finally resuspended in a volume of 2,500 μL of PBS. Finally, the comet assay was carried out as described above.
9
ATAC-seq from Frozen Pupae
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ATAC–seq was performed as previously described43 (link) with the following changes. The starting material was flash-frozen pupae. After thawing, whole pupae were homogenized in cold PBS and passed through a cell strainer (Corning, 352235). The cell suspension was counted, and 50,000 cells were used for each reaction. We used 250 ng of Tn5 prepared by the IMB Protein Production Core Facility per reaction and 15 PCR cycles in the library amplification step. Pooled samples were sequenced on NextSeq 500 High Output, PE for 2×75 cycles plus 2×8 cycles for the dual index read.
10
Evaluating STHB Formulation Stability
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To determine the stability and swelling ratio of STHB formulations, one gram of each formulation was placed in a cell strainer (Corning, Corning, NY) (n = 3). Each strainer was submerged in 7 mL of PBS on 6-well plates (Corning, Corning, NY) and incubated at 37 °C. Stability and swelling were recorded at 3, 7, 14, and 21 days by quantifying the wet weight and dry weight after lyophilization of STHB compositions. Degradation kinetics of the hydrogel formulations were calculated with the formula: mass loss percentage = (M0 – Md) / M0 × 100% [14 (link)]. M0 represents the original dry mass of the hydrogel, and Md is the mass of the hydrogel in the dry state after PBS incubation. Swelling ratio was calculated using the following formula: swelling ratio (Q) = (Ws – Wd) / Wd [14 (link)]. Ws represents the mass of the hydrogel after PBS incubation, and Wd is the original mass of the hydrogel.
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