40 m nylon cell strainer

Manufactured by BD

Sourced in United States

The 40 μm nylon cell strainer is a laboratory filtration device used to separate cells or particles based on size. It features a nylon mesh with a pore size of 40 microns, allowing for the effective separation of cells or other materials smaller than 40 μm.

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Cell strainer (717 citations) Nylon cell strainer (177 citations) 40-µm cell strainer (158 citations) 40 µm strainer (14 citations) 40 µm nylon strainer (2 citations) Nylon strainers (2 citations) Nylons (2 citations)

12 protocols using 40 m nylon cell strainer

Integrin Expression and Ligand Binding Assay

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Cells were harvested in PBS-EDTA by scraping and passed through a 40 µm nylon cell strainer (BD Falcon, Heidelberg, Germany) to yield single cells. After washing in FACS buffer (PBS, 5 mM EDTA and 3% FCS) the cells were stained. For the analysis of integrin expression, cells were incubated for 15 min in 100 µl FACS buffer with the following integrin antibodies (1:100 each): anti CD62-FITC (integrin β3) clone VI-PL2 (#336403); FITC mouse IgG control (#400109) (Bio Legend, Fell, Germany); anti-mouse CD29-FITC (integrin β1, # MCA2298F); anti-active integrin β1 12G10 (Alexa Fluor^® 488, ab202641); or hamster IgG negative control-FITC (# MCA2356F) (Bio-Rad AbD Serotec).
Cyclo-RGD-5-FAM (#65160) (AnaSpec, MoBiTech, Göttingen, Germany) was used to assess integrin ligand-binding activity. The RGD peptide represents the integrin-binding motif in fibronectin and vitronectin. Active integrins can therefore bind to the fluorescently labelled peptide and be quantified. Collected cells were washed in FACS buffer and subsequently stained with 2.5 µM Cyclo-RGD-5-FAM in 200 µl FACS buffer for 15 min before analysis.

Haun F., Neumann S., Peintner L., Wieland K., Habicht J., Schwan C., Østevold K., Koczorowska M.M., Biniossek M., Kist M., Busch H., Boerries M., Davis R.J., Maurer U., Schilling O., Aktories K, & Borner C. (2018). Identification of a novel anoikis signalling pathway using the fungal virulence factor gliotoxin. Nature Communications, 9, 3524.

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Isolation of Lymphocytes from Vaginal and Endometrial Tissue

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Tissue segments were obtained as either vaginal or endometrial surgical
samples after pelvic reconstructive surgery. Lymphocytes were isolated by
dissecting and mincing tissue with a scalpel into the smallest possible
fragments. Fragments were transferred to 50 ml conical tubes containing 30 ml of
warm 0.05% collagenase (Sigma type II collagenase #C6885) in
7.5% FBS RPMI media and incubated for 15 min at 37°C. Partially
digested tissue was mechanically disrupted 2× using a 16g blunt needle
attached to a 20ml syringe. Tissue was incubated for an additional 15 min at
37°C. Cell suspension was passed through a 40 µM nylon cell
strainer (BD Falcon, Franklin Lakes, NJ) and RPMI + 15% FBS (R15) was
added to quench enzyme activity. Cell suspension was pelleted 2× at 300
× g for 10 min at RT. After the second wash, the cell pellet was
resuspended in 5 ml of R15, and placed at 37°C with a loose cap.
Collagenase digestion was repeated 2×. All 3 pellet fractions were
combined and resuspended in 50 ml R15 and pelleted at 300 × g for 10
min. Supernatant was discarded and the remaining cell suspension was resuspended
in 1–3 ml of RPMI containing 10% AB serum. Cells were counted
and used for the various assays.

Moylan D.C., Goepfert P.A., Kempf M.C., Saag M.S., Richter H.E., Mestecky J, & Sabbaj S. (2017). Diminished CD103 (αEβ7) Expression on Resident T Cells from the Female Genital Tract of HIV-Positive Women. Pathogens & Immunity, 1(2), 371-389.

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Cell Cycle Analysis of PV-10 Treatment

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To analyze cell cycle alterations, the cells were seeded in 100 mm dishes (Corning Incorporated), so that a minimum of 2×10⁶ cells could be collected posttreatment. The cells were cultured for 24 hours, treated with either PBS (vehicle control) or PV-10 and cultured for either 16 or 24 hours, protected from light. Then they were collected by trypsinization, washed with PBS, filtered through a 40 µm nylon cell strainer (Falcon, Corning, NY, USA), counted by trypan blue staining using a hemocytometer, re-suspended in 0.9% (w/v) sterile NaCl, and fixed in ice-cold 90% (v/v) ethanol. Samples were incubated at room temperature for 30 minutes then stored at -20°C. For analysis, samples were centrifuged at 1,400 rpm for 5 minutes at 4°C and washed twice with ice-cold PBS. The cells were then incubated at 37°C for 20 minutes in 300 µL labeling buffer: 10 µg/mL DAPI (Sigma-Aldrich Co., St Louis, MO, USA), 200 µg/mL RNase A (Sigma-Aldrich Co.) in 0.1% Triton X-100 in PBS. The samples were run on a BD Bioscience LSR II cytometer using Diva 6.1.3 software (BD Bioscience, Mississauga, ON, Canada). The results were analyzed using ModFitLT 3.3 software (Verity Software House, Topsham, ME, USA).

Swift L., Zhang C., Trippett T, & Narendran A. (2019). Potent in vitro and xenograft antitumor activity of a novel agent, PV-10, against relapsed and refractory neuroblastoma. OncoTargets and therapy, 12, 1293-1307.

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Splenocyte Isolation Protocol

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Splenocytes were isolated in a cold RPMI+ medium. The spleen was put in a 40 µm nylon cell strainer (Falcon, Corning, NY, USA) in a Petri dish with a few ml of medium. Spleen was gently crushed using a syringe plunger until only connective tissue was left. The cell suspension was centrifuged at 450× g for 5 min at 4 °C, and red blood cell lysis was performed using 1× PharmLyse Buffer (BD Biosciences, San Jose, CA, USA) and washed twice with DPBS (Gibco, Bleiswijk, The Netherlands).

Jazbec K., Jež M., Švajger U., Smrekar B., Miceska S., Rajčevič U., Justin M., Završnik J., Malovrh T., Švara T., Gombač M., Ramšak Ž, & Rožman P. (2022). The Influence of Heterochronic Non-Myeloablative Bone Marrow Transplantation on the Immune System, Frailty, General Health, and Longevity of Aged Murine Recipients. Biomolecules, 12(4), 595.

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Immune Response Evaluation in Mice

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One week after the last vaccine dose, we euthanized mice and removed the spleen and lungs to assess immune responses. Single-cell suspensions of splenocytes were prepared by gently pressing the cells out of the spleen sac; lysing red blood cells with PharmLyse (BD Pharmingen); washing the cells; and filtering through a 70 µm nylon cell strainer (Falcon). Single-cell suspensions of lung cells were prepared by cutting the lung into small pieces with a scalpel; incubating at 37°C for 1 h with shaking in 10 mL of digestion solution (300 U/mL collagenase type II [Worthington] and 0.15 mg/mL DNase I [Worthington] in PBS); filtering through a 40-µm nylon cell strainer (Falcon); lysing red blood cells with PharmLyse (BD Pharmingen); and washing the cells. Advanced RPMI-1640 (Invitrogen) supplemented with 2% heat-inactivated fetal bovine serum, 2 mM glutamine dipeptide (glutaGRO Supplement, Corning), 10 mM HEPES buffer, 50 µM β-mercaptoethanol, and penicillin (100 IU/mL)-streptomycin (100 µg/mL) was used as the medium.

Tullius M.V., Bowen R.A., Back P.S., Masleša-Galić S., Nava S, & Horwitz M.A. (2024). LVS ΔcapB-vectored multiantigenic melioidosis vaccines protect against lethal respiratory Burkholderia pseudomallei challenge in highly sensitive BALB/c mice. mBio, 15(4), e00186-24.

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Bone Marrow Cell Isolation Protocol

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As previously reported by [18 (link),20 (link)], the mice were sacrificed using CO₂ asphyxiation and cervical dislocation, and the main bones (femurs, tibias, humeri, ilia, and spine) were dissected and thoroughly cleaned. Bones were crushed in cold RPMI+ medium (RPMI-1640 medium (Gibco, Bleiswijk, The Netherlands), supplemented with 25 mM HEPES (Gibco, Bleiswijk, The Netherlands), 300 mg/L L-glutamine (PAA, Cambridge, UK), 1 mM EDTA (Gibco, Bleiswijk, The Netherlands), and 1× PenStrep (Gibco, Bleiswijk, The Netherlands), using a sterile mortar and pestle. The cell suspension was filtered through a 40 µm nylon cell strainer (Falcon, Corning, NY, USA) twice and centrifuged for 5 min at 490× g at 4 °C. Red blood cell lysis was performed using 1× PharmLyse Buffer (BD Biosciences, San Jose, CA, USA) and washed twice with DPBS (Gibco, Bleiswijk, The Netherlands).

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Long-term Hematopoietic Reconstitution Analysis

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Egg incubation was prolonged until hatching and growing chicken were analyzed at 5 months post-transplantation to test long-term reconstitution potential. The peripheral blood was collected from the brachial vein into 1.3 ml collection tube containing EDTA. Cells were washed in PBS/FCS and stained with antibodies for 20 min at 4°C (antibodies are listed in Table S1). Multilineage reconstitution was analyzed in hematopoietic organs (thymus, spleen, bursa of Fabricius and BM). Organs were collected, crushed and cell suspensions were filtered (40 µm nylon cell strainer, Falcon) and washed twice in PBS/FCS. Single-cell suspensions were then stained (Table S1). 7-AAD was used to exclude dead cells. Flow cytometry analyses were performed on a FACSCalibur. Data were analyzed using FlowJo software.

Yvernogeau L, & Robin C. (2017). Restricted intra-embryonic origin of bona fide hematopoietic stem cells in the chicken. Development (Cambridge, England), 144(13), 2352-2363.

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Isolation and Analysis of Regulatory B Cells

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Splenocytes were isolated using a 40-µm nylon cell strainer (BD Biosciences, San Jose, CA, USA); RBCs were lysed with buffer containing 0.14 NH₄Cl and 0.017 M Tris-base (pH 7.5). IL-10-producing CD1d^highCD5⁺CD19⁺ Bregs were analyzed by flow cytometry after immunostaining of surface markers with the following monoclonal antibodies: fluorescein isothiocyanate (FITC)-conjugated anti-CD19 (clone 1D3) (BD Biosciences, San Jose, CA, USA), phycoerythrin (PE)-conjugated anti-CD1d (clone 1B1; isotype, Rat IgG2b, κ) (BD Biosciences), allophycocyanin (APC)-conjugated anti-CD5 (clone 53–7.3) (eBioscience, San Diego, CA, USA), and peridinin chlorophyll-protein complex (PerCP)- or PE-conjugated anti-IL-10 (clone JES5–16E3) (eBioscience). For subsequent intracellular IL-10 staining, the Cytofix/Cytoperm kit (BD Biosciences) was used according to the manufacturer's protocol (BD Biosciences). Intracellular transport processes were inhibited with BD GolgiStop containing the transport inhibitor monesin and Fc receptors were blocked by treatment with rat anti-mouse CD16/32 antibody (BD Bioscience) for 15 min. The FlowJo software (Tree Star Inc., Ashland, OR, USA)was used to analyze the flow cytometry data. To determine background staining, non-reactive isotype-matched control monoclonal antibodies (eBioscience) were used and cells were gated to exclude ≥98% of non-reactive cells.

Jeong Y.I., Hong S.H., Cho S.H., Park M.Y, & Lee S.E. (2016). Induction of IL-10-producing regulatory B cells following Toxoplasma gondii infection is important to the cyst formation. Biochemistry and Biophysics Reports, 7, 91-97.

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Murine Lymph Node Cell Isolation

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Preparation of single immune cell suspensions of mice lymph nodes used a standard protocol. Briefly, lymph nodes were crushed with PBS using a 40 µm nylon cell strainer (BD Biosciences, ON, Canada). The single-cell suspensions were centrifuged and resuspended in iced PBS buffer and stained with fluorescent-conjugated antibodies (BD Biosciences, Mississauga, ON, Canada) for surface markers: marking viability, APC-Cy7 anti-CD45, V450 anti-CD11b, PerCPCy5.5 anti-CD19, APC anti-CD3, FITC anti-CD4, PE anti-CD8. Polybead microspheres of 15 µm diameter (Polyscience, Inc., Warrington, PA, USA) were added to each sample before flow cytometry analysis (BD LSR II flow cytometer, BD Biosciences, ON, CAN). Data analysis was compiled using FlowJo software (BD Biosciences, Mississauga, ON, Canada).

Zhao Y., Aoudjit F, & Bourgoin S.G. (2022). Phospholipase A1 Member A Deficiency Alleviates Mannan-Induced Psoriatic Arthritis in Mice Model. International Journal of Molecular Sciences, 23(15), 8559.

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Isolation and Culture of Murine Cortical Neurons

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We used ICR mice in this study, in accordance with the Guidelines of the Asan Institute for Life Sciences for the Care and Use of Laboratory Animals. Cerebral cortical tissues were dissected from the brains of fetal ICR mice (Koatech, Pyeongtaek, Korea) at embryonic day E14, dissociated in Ca²⁺/Mg²⁺-free Hank’s balanced salt solution (HBSS; Invitrogen, Carlsbad, CA, USA) containing 0.25% trypsin-EDTA (Invitrogen), and filtered through 40-µm nylon cell strainer (BD Biosciences, Durham, NC, USA). Cells were washed in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and penicillin/streptomycin (Invitrogen), and resuspended in serum-free Neurobasal medium (Invitrogen) containing the B27 supplement (Invitrogen), L-glutamine (2 mM; Invitrogen) and penicillin/streptomycin. Cells were plated at a density of 5 × 10⁵–10⁶ cells/well on poly-L-lysine-coated well culture dishes and grown in a humidified 5% CO₂ incubator at 37°C. Cultures were treated with cytosine arabinoside (Ara-C, 2 µM; Sigma, St. Louis, MO, USA) for 24 h at 3 days in vitro (DIV3) to halt the growth of non-neuronal cells, and maintained in fresh Neurobasal medium with B27 until used in experiments between DIV10–11.

Kim S., Seo J.W., Oh S.B., Kim S.H., Kim I., Suh N, & Lee J.Y. (2015). Disparate roles of zinc in chemical hypoxia-induced neuronal death. Frontiers in Cellular Neuroscience, 9, 1.

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