40 μm nylon strainer

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The 40-μm nylon strainer is a laboratory filtration device designed to separate particles larger than 40 micrometers from a liquid or suspension. It is made of nylon material and has a pore size of 40 micrometers.

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8 protocols using 40 μm nylon strainer

Isolation and Characterization of Rat ADSC

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Rat ADSC was extracted from inguinal adipose tissue of Lewis rats (4 to 6 weeks old). These samples were first digested by 0.1% collagenase (NB4, Serva, France) for 1 h under gentle agitation at 37 °C, before being filtered through a 40-μm nylon strainer (Falcon, Corning, USA). The resultant substrate was subjected to centrifugation before the cell pellets were resuspended in 10 cm² culture plates at 37 °C, under a 5% CO₂ atmosphere which contained low glucose complete Dulbecco’s Modified Eagle Medium supplemented with 100 U/mL penicillin and 100 mg/mL streptomycin (Gibco, Invitrogen, USA) and 10% fetal bovine serum (FBS; Sciencell, USA). Further studies utilized cell passages 2 through 4.
Flow cytometric analysis of the cell surface marker profile of ADSC was performed with a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA) using primary antibodies for CD 90, CD 45, and CD44 (BioLegend Inc., San Diego, USA). A differentiation medium (Cyagen Bioscience, Inc., Guangzhou, China) was used to induce adipogenesis, osteogenesis, and chondrogenesis. Oil red, alizarin red, and alcian blue stains were used to identify differentiated cells.

Chen J., Wang Y., Hu H., Xiong Y., Wang S, & Yang J. (2021). Adipose-derived cellular therapies prolong graft survival in an allogenic hind limb transplantation model. Stem Cell Research & Therapy, 12, 94.

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Isolation and Purification of Muscle-Derived Cells

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Muscle tissue was washed in 1× PBS, cleaned of any visible fat depositions and finely minced. Then, 2 g of the minced tissue was transferred to digestion buffer 1 (750 U ml⁻¹ collagenase type 2 in 1× PBS) and incubated at 37 °C in a water bath for 90 min. The partially digested tissue was collected by centrifugation (650g, 5 min, 4 °C) and the pellet was resuspended in digestion buffer 2 (100 U ml⁻¹ collagenase type 2, 2 U ml⁻¹ dispase in PBS). After 30 min incubation at 37 °C in a water bath, the digestion was stopped by the addition of 2% FBS. Cells were filtered through a 100-μm and a 40-μm nylon strainer (BD Falcon), collected by centrifugation (650g, 4 °C, 3 min) and washed with 1× PBS, 2% FBS. Subsequently, a 20% Percoll gradient (15,000g, 4 °C, 20 min) was used for cell purification. The layer containing cells was collected, washed in PBS containing 2% FBS, and viable cells were counted by Trypan Blue exclusion using a haemocytometer. Nuclei were profiled using a Chromium Controller (10X Genomics) according to the manufacturer’s protocol.
The methods key resources table is in Supplementary Table 24.

Litviňuková M., Talavera-López C., Maatz H., Reichart D., Worth C.L., Lindberg E.L., Kanda M., Polanski K., Heinig M., Lee M., Nadelmann E.R., Roberts K., Tuck L., Fasouli E.S., DeLaughter D.M., McDonough B., Wakimoto H., Gorham J.M., Samari S., Mahbubani K.T., Saeb-Parsy K., Patone G., Boyle J.J., Zhang H., Zhang H., Viveiros A., Oudit G.Y., Bayraktar O.A., Seidman J.G., Seidman C.E., Noseda M., Hubner N, & Teichmann S.A. (2020). Cells of the adult human heart. Nature, 588(7838), 466-472.

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Establishment of Camel Fibroblast Cell Lines

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Fibroblast cell lines were established from the ear skin of an elite camel; samples were obtained as previously described with minor modifications (Wani et al., 2010) . Briefly, tissues were washed three times with DPBS supplemented with 1% antibiotic-antimycotic. After that, samples were minced into small pieces with a surgical blade and digested at 38°C in a humidified atmosphere with 5% CO 2 for 2 h in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific) supplemented with 0.1% collagenase type II (Thermo Fisher Scientific). The dispersed cells were washed with DPBS by centrifugation at 300 g for 5 min and filtered through a 40-μm nylon strainer (Falcon, Franklin, NJ, USA). The cell pellets were cultured at 38°C in a humidified atmosphere with 5% CO 2 in DMEM supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific), 1% nonessential amino acids (Thermo Fisher Scientific), 1% antibiotic-antimycotic (Thermo Fisher Scientific), and 0.1% β-mercaptoethanol (Thermo Fisher Scientific). The culture medium was changed every 2 days until confluency reached 80%, and then the cells were passaged using 0.25% trypsin-EDTA solution.

Son Y.B., Yu X., Jeong Y.I., Hossein M.S., Olsson P.O., Jeong Y.W., Choi E.J., Tinson A.H., Singh K.K., Rajesh S., Noura A.S, & Hwang W.S. (2022). Comparison of pregnancy rate in dromedary camel between early-stage embryos and blastocyst transfer produced by somatic cell nuclear transfer using in vitro-matured oocytes. Zygote (Cambridge, England), 30(4).

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Immunophenotypic Characterization of hWJSCs

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Cultured hWJSC monolayers were first disassociated using trypsin (TrypLE™ Express, Thermo Scientific) for 3–5 min at 37 °C in a 5% CO₂ in air, then centrifuged and washed in phosphate buffered saline (PBS) and blocked with 10% normal goat serum (NGS) (Thermo Scientific) for 10 min at room temperature to prevent non-specific binding following manufacturer’s instruction. The cells were then incubated with mouse monoclonal primary antibodies for a series of CD markers viz., CD14, CD19, CD29, CD34, CD44, CD45, CD73, CD90, CD105, HLA-ABC, and HLA-DR (1:100) (Biolegend, San Diego, CA) for 30 min at 4 °C. This was followed by incubation with Alexa Fluor®488 (1:5000) goat anti-mouse secondary antibody (Thermo Scientific) for 30 min at 4 °C in the dark [29 (link)]. The cells were finally washed in PBS (−), re-suspended in 10% NGS, and filtered using a 40-μm nylon strainer (BD) to remove any cell clumps and then analyzed using a CyAn™ ADP Analyzer (Beckman Coulter, Fullerton, CA).

Lin H.D., Fong C.Y., Biswas A, & Bongso A. (2020). Allogeneic human umbilical cord Wharton’s jelly stem cells increase several-fold the expansion of human cord blood CD34+ cells both in vitro and in vivo. Stem Cell Research & Therapy, 11, 527.

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Isolation and Identification of Mouse ILCs

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Splenic cells were isolated by mashing spleen through a 40 μm Nylon strainer (BD Biosciences). Red blood cells were removed using ACK lysis buffer (150 mM NH₄Cl, 1 mM KHCO₃, 0.1 mM Na₂EDTA). Single cell suspensions of splenic cells were resuspended in AutoMACS running buffer containing biotin-conjugated anti-mouse Lineage cocktail plus biotin-conjugated anti-mouse NK1.1 and F4/80, and incubated on ice for 20 min. After centrifuge the supernatant was discarded and the splenic cells resuspended in AutoMACS running buffer plus Streptavidin Microbeads (Miltenyi Biotec). Lineage-negative cells were purified using an AutoMACS Separator Pro system (Miltenyi Biotec) and stained with anti-mouse CD45 Microbeads (Milteny Biotec). Lineage-negative, CD45-positive cells were deemed to constitute the ILC subset. Purified innate lymphoid cells (ILCs) were stained with antibodies to CD45.2 (104; 0.5 μg/ml), Thy1.2 (30-H12; 2 μl for 1*10⁶ cells), CD127 (A019D5; 5 μg/ml) antibody and FITC-conjugated streptavidin, and were acquired on a FACSFortessa instrument (BD Biosciences) and analyzed using the FlowJo cytometric analysis program (Tree Star).

Yang F.C., Chiu P.Y., Chen Y., Mak T.W, & Chen N.J. (2019). TREM-1-dependent M1 macrophage polarization restores intestinal epithelium damaged by DSS-induced colitis by activating IL-22-producing innate lymphoid cells. Journal of Biomedical Science, 26, 46.

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Chondrogenic Induction Pellet Characterization

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After 1, 3, or 7 days of chondrogenic induction with TGFβ1 or the combination of TGFβ1 and BMP7, pellets were collected and washed with ice-cold PBS and fixed with 2% formaldehyde (Polysciences, Warrington, PA). Pellets were then dissociated using collagenase A and mechanical disruption (Roche Mannheim, Germany). Cells were passed through a 40-μm nylon strainer (BD Biosciences), washed with a buffer solution, and incubated with antibodies detecting TGFβR2, BMPR1B and BMPR2. The data is presented graphically as the fluorescence signal to noise ratio against the geometric mean of the isotype control.

Lee P.T, & Li W.J. (2016). Chondrogenesis of Embryonic Stem Cell-derived Mesenchymal Stem Cells Induced by TGFβ1 and BMP7 Through Increased TGFβ Receptor Expression and Endogenous TGFβ1 Production. Journal of cellular biochemistry, 118(1), 172-181.

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Phenotyping Stem Cells under Hypoxia

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The cells grown under normoxic (21% O₂) and hypoxic (10% or 5% O₂) conditions were first collected using trypsin (TrypLE™ Express, Thermo Scientific) for 3-5 mins at 37°C in a 5% CO₂ in air, washed in phosphate-buffered saline (PBS), and blocked with 10% normal goat serum (NGS) (Thermo Scientific) for 10 mins to prevent nonspecific binding. The cells were then incubated with mouse monoclonal primary antibodies for a series of CD markers—CD14, CD19, CD24, CD29, CD34, CD40, CD44, CD45, CD49d, CD73, CD90, CD105, CD117, CD140b, CD146, CD271, HLA-DR, and CD108-PE (1 : 100) (BioLegend, San Diego, CA)—for 30 mins. This was followed by incubation with Alexa Fluor®488 (1 : 500) goat anti-mouse secondary antibody (Thermo Scientific) for 30 mins. The cells were finally washed in PBS, resuspended in 10% NGS, filtered using a 40 μm nylon strainer (BD) to remove any cell clumps, and analyzed using a CyAn™ ADP Analyzer (Beckman Coulter, Fullerton, CA).

Lin H.D., Fong C.Y., Biswas A, & Bongso A. (2020). Hypoxic Wharton's Jelly Stem Cell Conditioned Medium Induces Immunogenic Cell Death in Lymphoma Cells. Stem Cells International, 2020, 4670948.

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Isolation of Adipose Stromal Cells from Epididymal Fat

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Epididymal adipose tissue was washed with 1 × phosphate buffer saline (Life Technologies Inc., Burlington, ON, Canada) and visible blood vessels were removed from the fat pads. The minced tissues were digested for 1 h at 37 °C with type II collagenase solution (1.2 mg ml⁻¹; Sigma-Aldrich Canada Ltd) and 2% bovine serum albumin fraction V (Roche Applied Science, Laval, QC, Canada) in phosphate buffer saline (pH 7.4) in an orbital shaking oven. Cell suspensions were centrifuged in polyethylene centrifuge tubes (Dow Corning Corporation, Midland, MI, USA) for 5 min at 250 × g and the floating layer composed of adipocytes was discarded. The pellet was resuspended in 5 ml erythrocyte lysis buffer (8.26 g NH₄Cl, 1 g KHCO₃ and 0.037 g EDTA in 1 liter water, pH 7.3; Sigma-Aldrich Canada Ltd) at room temperature for 5 min, followed by the addition of phosphate buffer saline (pH 7.4) to stop the lysis reaction. Cells were initially filtered through 100-μm nylon strainer and, subsequently, a 40-μm nylon strainer (BD Canada, Mississauga, ON, Canada). Finally, cell suspensions were centrifuged at 350 × g for 5 min at 4 °C and the pellet was stored at −80 °C until RNA extraction.

Campioli E., Martinez-Arguelles D.B, & Papadopoulos V. (2014). In utero exposure to the endocrine disruptor di-(2-ethylhexyl) phthalate promotes local adipose and systemic inflammation in adult male offspring. Nutrition & Diabetes, 4(5), e115-.

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