Left and right femurs and muscle tissues surrounding those structures of 14.5 to 19.5 dpc C57BL/6 mouse embryos were used to obtain single cell suspensions. Tissues were trimmed from femurs and incubated with 3 mg/ml collagenase in medium containing 10% fetal bovine serum (FBS) for 20 minutes at 37°C. Cells were then filtered through 70 μm nylon cell strainers (BD Biosciences, San Jose, CA). BM cells were flushed out with PBS containing 2% FBS using 29 to 32G needles with syringes (TERUMO, Tokyo, Japan) and filtered through 40 μm nylon cell strainers (BD Biosciences). FLs from 14.5 dpc and 16.5 dpc embryos were dissected out, and single cell suspensions were prepared by digesting tissues with 3 mg/ml collagenase in medium containing 10% FBS for 20 minutes at 37°C. Cells were then filtered through 40 μm nylon cell strainers (BD Biosciences). BM cells from femurs and tibias of 3-month-old adult C57BL/6 mice were dissected out and then flushed out with PBS containing 2% FBS from 27G needles and syringes (TERUMO). Cells were then filtered through 40 μm nylon cell strainers (BD Biosciences). Fetal blood was obtained from 16.5 dpc embryos. Blood cells were then washed 3 times with PBS containing 2% FBS.
40 μm nylon cell strainer
Manufactured by BD
Sourced in United States
The 40-μm nylon cell strainer is a laboratory tool used for filtering and separating cells from a cell suspension. It is designed to retain cells larger than 40 micrometers while allowing smaller particles to pass through.
Automatically generated - may contain errors
Lab products found in correlation
70 μm nylon cell strainer, by BD (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Facsaria 2 flow cytometer, by BD (3 mentions)
Penicillin streptomycin, by Merck Group (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Iscove s modified eagle medium imdm, by Thermo Fisher Scientific (2 mentions)
100 μm nylon cell strainer, by BD (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
M csf, by R&D Systems (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Hanks balanced salt solution (hbss), by Merck Group (2 mentions)
Athymic nude mice, by Charles River Laboratories (2 mentions)
Collagenase type 3, by Worthington (2 mentions)
72 protocols using 40 μm nylon cell strainer
1
Isolation of Murine Embryonic and Adult Cells
2
Isolation and Preparation of Splenic Cells
3
Isolation and Characterization of Splenocytes
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Spleens were isolated aseptically and single-cell suspensions made in complete media comprising Iscove’s Modified Eagle Medium (IMDM) (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 100 U/mL penicillin G and 100 μg/mL streptomycin. Single-cell suspensions were achieved by passing the organs through a 40-μm nylon cell strainer (Becton Dickson, NJ) using a 2-mL syringe plunger. Cells were then spun at 1200 rpm for 5 min and media discarded, and the red blood cells lysed by resuspending in 1 mL RBC lysis buffer (8.34 g ammonium chloride, 0.037 g EDTA and 1 g sodium hydrogen carbonate/L, pH 7.2) for 1 min. Cells were pelleted again and resuspended in complete media. Viability was determined by trypan blue exclusion. Cells were then reconstituted to a working concentration of 107 cells/mL and used for culture and flow cytometry. Cells were plated in a 96-well plate and stained for expression of extracellular markers.
4
Primary neuron and glial cell extraction from fetal rat brains
5
Murine Thymocyte Isolation and Apoptosis
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Following cervical dislocation thymi were dissected from 4–8 week old male Balb/c mice. Thymocytes were dissociated by grinding thymi through a cell strainer (40μM nylon, BD Falcon). Thymocytes were used immediately or aged overnight in RPMI 1640 at 37°C and 5% CO2 to generate a cell population with variable levels of apoptosis. Following incubation cells were centrifuged at 230 x g for 6-minutes, and washed with PBS.
6
Embryo Extraction and Protein Quantification
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Sixty females and 10 males were incubated together inside embryo collectors with embryo dishes for a certain number of hours according to the desired stage of embryos to be harvested. Collected embryos were gently rinsed off the medium with embryo collection buffer (Triton X-100 0.03%; NaCl 68 mM). Embryos were removed from the medium using a brush and poured into a sieve (Falcon Cell Strainer 40 μM Nylon, 352340). Harvested embryos were washed again and collected in a fresh tube (up to 50 μl of embryos corresponding to 100 embryos). Laemmli 2× was added in ratio 1:1 in comparison to harvested volume and embryos were lysed by means of a pestle. After boiling embryo’s mush at 95°C for 5 min, chorion residues were removed by centrifugation with a benchtop centrifuge (maximum speed) at room temperature. Protein concentration was estimated by Amido Black staining using bovine serum albumin of known concentration as a reference.
7
Sequential Filtration of Seed Cultures
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Seed cultures were grown for 24, 48, and 72 h before being sequentially filtered through 100, 40, and 5 μm filters (Falcon® Cell Strainer 100 μm Nylon, Falcon® Cell Strainer 40 μm Nylon, PluriSelect pluriStrainer® 5 μm). The 40 μm filtration step was necessary to prevent clogging of the 5 μm filter. The 48- and 72-h samples were inspected without further preparation, whereas for the 24-h sample 15 ml of the filtrate were concentrated via centrifugation at 1000 rpm for 30 min at 4°C and subsequently analyzed.
8
Hippocampal Tissue Isolation for Flow Cytometry
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On either day 4 or day 5 post MWM training, mice were euthanized with halothane to collect brain tissue samples. The hippocampus was isolated according to a modified protocol following transcardial perfusions34 (link) and collected into CentriStar cap 15 ml Corning centrifuge tubes (Corning, NY) in Isove’s Modified Dulbecco’s Medium (IMDM) (GIBCO/Invitrogen; Carlsbad, CA), 10% Fetal Calf Serum (FCS), and penicillin streptomycin (P/S) on ice. Tissue was pushed through 40 μm nylon cell strainers (Falcon, Corning Incorporated, NY) and centrifuged at 1200 rpm at 4 °C for 10 min to be used for flow cytometry. Other samples were snap frozen for qPCR.
9
Hippocampus Isolation and Analysis
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On day 8 post infection, mice were euthanized with halothane to collect hippocampi. Hippocampi were collected into CentriStar™ cap 15 ml Corning® centrifuge tubes (Corning, NY) in Isove’s Modified Dulbecco’s Medium (IMDM) (GIBCO/Invitrogen; Carlsbad, CA), 10% Fetal Calf Serum (FCS), and penicillin streptomycin (P/S) on ice. Tissue was pushed through 40 μm nylon cell strainers (Falcon®, Corning Incorporated, NY), centrifuged at 1200 rmp at 4 °C for 10 min and re-suspended in 450 μl IMDM buffer for flow cytometry staining.
10
Cell Diameter Measurement by Imaging Flow Cytometry
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One million cells were plated in 150-mm dishes, incubated for 24 h, and harvested by trypsinization. Cells were then washed with PBS and fixed with 2% PFA in 0.1 M sodium phosphate buffer for 20 min at room temperature. Samples were washed to remove fixative and filtered using 40-μm nylon cell strainers (BD Falcon, Durham, NC). For analysis, cells were suspended to a final volume of 100 μl in PBS. To determine cell diameter, bright-field images of individual cells were acquired using an Amnis ImageStreamXmkII imaging flow cytometer and analyzed with INSPIRE software. More than 1800 cells were measured per genotype per experiment.
For cell diameter measurement of SMCR8 siRNA/torin1–treated cells, 500,000 WT HeLa M cells were transfected with control or SMCR8 siRNA and plated in a 150-mm dish. After 24 h, 200 nM torin1 was added to cells receiving this treatment, followed by a 48-h incubation. Cells were then prepared for cell diameter measurement as described.
For cell diameter measurement of SMCR8 siRNA/torin1–treated cells, 500,000 WT HeLa M cells were transfected with control or SMCR8 siRNA and plated in a 150-mm dish. After 24 h, 200 nM torin1 was added to cells receiving this treatment, followed by a 48-h incubation. Cells were then prepared for cell diameter measurement as described.
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