70 μm strainer

Manufactured by Thermo Fisher Scientific

Sourced in United States

The 70 μm strainer is a laboratory filtration device designed to remove particles or debris larger than 70 micrometers from liquid samples. It is a useful tool for sample preparation and processing in various scientific applications.

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Lab products found in correlation

25 protocols using 70 μm strainer

Isolation and Analysis of Thymic Epithelial Cells

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Single cell suspensions of TECs were obtained collagenase (Collagenase D type IV, Worthington) and dispase (Roche) enzymatic dissociation as previously described [78 (link)] and thymocytes were obtained by pressing the thymus through a 70μm strainer (Fisher). Cells were stained with fluorochrome-conjugated antibodies in FACS buffer (PBS pH 7.2, 0.005M EDTA, 2% FBS) for 20 minutes on ice and washed. Propidium iodide (Invitrogen) was added (0.5 μg/ml) to each sample prior to analysis to exclude dead cells. Anti-CD326 (clone G8.8), anti-I-A/I-E (clone M5/114.15.2), anti-CD25 (clone PC61) and anti-CD117 (clone 2 B8) were purchased from Biolegend. Anti-CD44 (clone IM7), anti-CD8α (clone 53–6.7), anti-CD45 (clone 30-F11) and anti-Bcl-2 (clone 3F11) were purchased from eBioscience. Biotinylated anti-Ly51, anti-pStat3 (clone 4/p-Stat3 pY705) and anti-total Stat3 (clone M59-50) were purchased from BD Biosciences. Anti-CD4 (clone RM4-5) and Streptavidin Qdot 655 was purchased from Invitrogen. To exclude erythrocytes, granulocytes, dendritic cells, macrophages and NK cells from the thymocyte analyses, the following antibodies were purchased from eBioscience: TER-119, CD11c (cloneN418), CD11b (clone M1/70), NK-1.1 (clone PK136) and Ly-6G (clone RB6-8C5). Flow cytometry was performed on a Becton Dickenson FACS Aria II and data were analyzed using FlowJo software (Tree Star).

Lomada D., Jain M., Bolner M., Reeh K.A., Kang R., Reddy M.C., DiGiovanni J, & Richie E.R. (2016). Stat3 Signaling Promotes Survival And Maintenance Of Medullary Thymic Epithelial Cells. PLoS Genetics, 12(1), e1005777.

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Isolation and Culture of Amnion Membrane-Derived Cells

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All reagents and media were warmed to 37°C prior to use. The amnion membrane was manually peeled from normal, term, not in labor cesarean section placentas, then rinsed in saline and transferred to a Petri dish that contained Hanks Balanced Salt Solution (HBSS) (Mediatech Inc., Manassas, VA). The amnion membrane was then cut into 2 cm x 2 cm pieces. They were digested twice in 0.25% trypsin and 0.125% collagenase A (Sigma-Aldrich, St Louis, MO) in HBSS for 35 minutes at 37°C. After each digestion, the tissue was filtered through a 70μm strainer (Fisher Scientific, Waltham, MA) cell and trypsin was inactivated using complete Dulbecco’s Modified Eagle’s Medium: Nutrient Mixture F-12 media (DMEM/F12) (Mediatech Inc.) supplemented with 15% fetal bovine serum (FBS) (Sigma-Aldrich), 10% penicillin/streptomycin, 10% amphotericin B (Mediatech Inc.), and 50μg/mL epidermal growth factor (EGF) (Sigma-Aldrich) The collected filtrate was centrifuged for 10 minutes at 3000 g. The cell pellet was re-suspended in 5 mL complete DMEM/F12. The cells were then counted using a hemocytometer. Once cells were counted, approximately 3-5 million cells per flask were cultured in T75 flasks containing complete DMEM/F12 media at 37°C, 5% CO₂, and 95% air humidity until they were 80-90% confluent.

Richardson L.S., Radnaa E., Urrabaz-Garza R., Lavu N, & Menon R. (2020). Stretch, Scratch, and Stress: Suppressors and Supporters of Senescence in Human Fetal Membranes. Placenta, 99, 27-34.

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Isolation and Analysis of Fetal Thymocytes

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For TEC analysis fetal thymi were digested with 0.125% Collagenase (Sigma) and 0.1% DNase (Roche) as previously described^{7 (link)}. For analysis of thymocyte subsets, E15.5 thymi were pressed through a 70 μm strainer (Fisher). Cells were stained with fluorochrome-conjugated antibodies in FACS buffer (PBS pH 7.2, 0.005 M EDTA, 2% FBS) for 15 minutes on ice and washed with FACS buffer. Propidium iodide (Invitrogen) was added (0.5 μg/ml) to each sample prior to analysis to exclude dead cells. Cells were stained with the following antibodies purchased from Biolgend or eBioscience: anti-CD326 (clone G8.8), anti-I-A/I-E (clone M5/114.15.2), biotinylated anti-Rat Ly51 (Clone 6C3), anti-CD25 (clone PC61), anti-CD117 (clone 2 B8), anti-CD44 (clone IM7), anti-CD8α (clone 53-6.7), anti-CD45 (clone 30-F11), anti-CD4 (clone RM4-5). To exclude erythrocytes, granulocytes, dendritic cells, macrophages and NK cells from the thymocyte analyses, we used the following antibodies, all conjugated to PE-Cy5 were purchased: TER-119, CD11c (cloneN418), CD11b (clone M1/70), NK-1.1 (clone PK136) and Ly-6G (clone RB6-8C5). Cells were analyzed or sorted on a FACS Aria II (BD Science). Data were analyzed using FlowJo software (Tree Star).

Singarapu N., Ma K., Reeh K.A., Shen J., Lancaster J.N., Yi S., Xie H., Orkin S.H., Manley N.R., Ehrlich L.I., Jiang N, & Richie E.R. (2018). Polycomb Repressive Complex 2 is essential for development and maintenance of a functional TEC compartment. Scientific Reports, 8, 14335.

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Isolation of Neutrophils from Ire1α-deficient Mice

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Mice bearing Ire1α floxed alleles (Ire1α^flox/flox) (34 (link), 35 (link)) were crossed with S100A8 promoter-driven Cre recombinase (MRP8-Cre) mice (36 (link)). The resulting MRP8-Cre⁺Ire1α^WT/flox mice were then backcrossed with Ire1α^flox/flox to generate mice deficient in neutrophil IRE1α (MRP8-Cre⁺Ire1α^flox/flox) and control WT littermates (MRP8-Cre⁻Ire1α^flox/flox). Bone marrow was extracted from mouse femurs and tibias and mechanically dissociated into single-cell suspensions using a 70 μm strainer (Fisher Scientific). Cells were collected by centrifugation and re-suspended in 2 ml HBSS. Neutrophils were isolated by Percoll gradient as previously described (37 (link)). Briefly, bone marrow cells were overlaid on three layers of Percoll (78%, 69% and 52%) and centrifuged at 1500g for 30 minutes without braking. Cells from the interface of 78% and 69% were collected, washed with PBS and centrifuged (1600 rpm, 5 min, 4°C). Pellets containing neutrophils were re-suspended in medium (RPMI + 10% FBS) and counted using an Invitrogen automated cell counter. Neutrophil purity was assessed by flow cytometry using anti-Ly-6G antibody (Biolegend).

Abuaita B.H., Sule G.J., Schultz T.L., Gao F., Knight J.S, & O’Riordan M.X. (2021). The IRE1α stress signaling axis is a key regulator of neutrophil antimicrobial effector function. Journal of immunology (Baltimore, Md. : 1950), 207(1), 210-220.

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Serum Isolation and Immune Cell Harvesting

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Blood was collected, and serum was clarified by collection of the supernatant following centrifugation at 10,000 rpm for 5 min. Two centrifugation steps were performed, and following clarification, serum was stored at −20°C for further analysis. Splenic and iliac lymph node (LN) mononuclear cells were harvested as previously described (26 (link)). Tissues were ground through a 70-μm strainer (Fisher Scientific), red blood cells were lysed by ammonium-chloride-potassium (ACK) treatment for 3 min, and debris was removed by subsequent filtering through a 30-μm filter (Miltenyi Biotec).

Provine N.M., Badamchi-Zadeh A., Bricault C.A., Penaloza-MacMaster P., Larocca R.A., Borducchi E.N., Seaman M.S, & Barouch D.H. (2016). Transient CD4+ T Cell Depletion Results in Delayed Development of Functional Vaccine-Elicited Antibody Responses. Journal of Virology, 90(9), 4278-4288.

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Expansion of Pmel-specific T cells

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Spleens from Pmel mice were mechanically disrupted into individual cells by smashing on a 70-μm strainer (Fisher Scientific, Pittsburgh, PA, USA) and then lysed with ACK lysing buffer (2 ml per spleen, Gibco/Thermo Fisher Scientific) for 5 min to get rid of the red blood cells. The cells were then washed with PBS and resuspended to a cell density of around 1.0 × 10⁶/ml with complete RPMI 1640 culture medium (Gibco/Thermo Fisher Scientific) containing FBS (10%, v/v), Hepes (1%, v/v), penicillin/streptomycin (1%, v/v), and β-mercaptoethanol (0.1%, v/v), supplemented with mouse IL-2 (10 ng/ml; PeproTech, London, UK), IL-7 (2 ng/ml; PeproTech), and gp100_25–33 (1 μM; GenScript, Piscataway, NJ, USA). After culturing for 3 days at 37°C with 5% CO₂, the live cells were enriched by density gradient centrifugation with Ficoll-Paque PLUS (GE Healthcare). The collected cells were cultured for extra 2 days with a cell density of around 1.0 × 10⁶/ml in complete RPMI medium supplemented with mouse IL-2 (10 ng/ml) and IL-7 (2 ng/ml) before use.

Zhao Y., Xie Y.Q., Van Herck S., Nassiri S., Gao M., Guo Y, & Tang L. (2021). Switchable immune modulator for tumor-specific activation of anticancer immunity. Science Advances, 7(37), eabg7291.

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Pineal Gland Single Cell Isolation

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At 24 h (P8) and 72 h (P10) post surgeries, neonatal rats were euthanized, and the pineal glands were immediately dissected out from their brains. The protocol of generating pineal single cell solution was adapted from previous publications (Schaad et al., 1993 (link); Mays et al., 2018 (link)). In brief, we first made and preheated (5% CO₂ at 37°C, 0.5 h) the digestion solution [20 U/ml papain (Sigma, 9001-73-4), dnase 100 mg/L (Sigma, DN-25), 0.2 U/ul SuperaseIn RNase Inhibitor (Thermofisher Scientific, AM2694) in 1x HBSS]. For each time point/condition, pineal glands from multiple animals were pooled and added with preheated papain solution, incubated at 37°C. During this period, we applied intermittent agitation in each 10 min by performing gentle titration using a 1 ml pipette tip. After 45 min, the digestion solution containing pineal cells were filtered by a pre-wetted 70 μm strainer (FisherScientific, 08-771-2) and centrifuged (300 g, 4 C) for 5 min. Pellets were then resuspended in 1xPBS (0.1% BSA) for single cell sequencing.

Ding X., Pan T., Tian Q., Huang W., Hayashi L.S., Liu Q., Li F., Xu L.X., Miao P., Yang X., Sun B., Feng C.X., Feng X., Li M, & Huang J. (2022). Profiling Temporal Changes of the Pineal Transcriptomes at Single Cell Level Upon Neonatal HIBD. Frontiers in Cell and Developmental Biology, 10, 794012.

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Tracking Adipose-Derived Stem Cell Homing

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Additional groups of mice (three for each condition) were used to assess the homing of injected ASCs. To do this, mice were infused with a single tail vein injection of 2 × 10⁵ cells or saline and were sacrificed 14 h later. Organs were then extracted and homing was examined in the peripheral blood, spleen, liver, lungs, scWAT and vWAT. Individual cells from the different organs were obtained after digestion in 0.1% collagenase (as described above) for 1 h and 30 min at 37 °C with gentle agitation. Digested tissues were passed through a 70-μm strainer (Fisher Scientific, Waltham, MA). In the case of peripheral blood, we used the 1 × Lyse lysing solution (BD Pharmingen, Oxford, UK) to lyse red blood cells. In total, 1 × 10⁷ cells were used for staining with a phycoerythrin-conjugated CD90 monoclonal antibody (Clone 5E10, BD Pharmingen) after preincubation with FcR blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells obtained from non-transplanted mice stained with the same antibodies were used to exclude false positive cells and to establish the negative control. Data were acquired on FACSAria III flow cytometer (BD Biosciences) and were analyzed using FACSDiva v8 (BD Biosciences) and FlowJo v10 (Ashland, OR) software.

Calvo E., Keiran N., Núñez-Roa C., Maymó-Masip E., Ejarque M., Sabadell-Basallote J., del Mar Rodríguez-Peña M., Ceperuelo-Mallafré V., Seco J., Benaiges E., Michalopoulou T., Jorba R., Vendrell J, & Fernández-Veledo S. (2021). Effects of stem cells from inducible brown adipose tissue on diet-induced obesity in mice. Scientific Reports, 11, 13923.

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Isolation of Adult Mouse Keratinocytes

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Adult (6–8 weeks) mouse keratinocytes were isolated from male or female animals using protocols described previously ^{78 (link)}. In brief, tail skin was incubated in 4mg/ml Dispase II (Roche) overnight at 4°C. The epidermis was physically removed from the dermis with tweezers and subsequently digested with TrypLE (Thermo) for 20 min at room temperature, and keratinocytes were detached by vigorously shaking in culture medium and filtered with a 70 μm strainer (Fisher scientific). Isolated cells were seeded at a density of 10⁵cells/cm² cultured with EpiLife (Gibco) in 12-well plates precoated with coating matrix (Gibco) at 37°C with 5% CO₂ and 100% humidity and used between 3 and 5 days after isolation.

Artsimovitch I., Svetlov V., Anthony L., Burgess R.R, & Landick R. (2001). RNA Polymerases from Bacillus subtilis and Escherichia coli Differ in Recognition of Regulatory Signals In Vitro. Journal of Bacteriology, 183(4), 1504.

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Isolation and Culture of Amniotic Epithelial Cells

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All reagents and media were warmed to 37°C prior to use. The amnion membrane was manually peeled from normal, term, not in labor cesarean section placentas, then rinsed in saline and transferred to a Petri dish that contained Hanks Balanced Salt Solution (HBSS) (Mediatech Inc., Manassas, VA, United States). The amnion membrane was then cut into 2 cm × 2 cm pieces. They were digested twice in 0.25% trypsin and 0.125% collagenase A (Sigma-Aldrich, St. Louis, MO, United States) in HBSS for 35 min at 37°C. After each digestion, the tissue was filtered through a 70 μm strainer (Fisher Scientific, Waltham, MA, United States) cell and trypsin was inactivated using complete Dulbecco’s Modified Eagle’s Medium: Nutrient Mixture F-12 media (DMEM/F12) (Mediatech Inc.) supplemented with 15% fetal bovine serum (FBS) (Sigma-Aldrich), 10% penicillin/streptomycin, 10% amphotericin B (Mediatech Inc.), and 50 μg/mL epidermal growth factor (EGF) (Sigma-Aldrich). The collected filtrate was centrifuged for 10 min at 3,000 g. The cell pellet was re-suspended in 5 mL complete DMEM/F12. The cells were then counted using a hemocytometer. Once cells were counted, approximately 3–5 million cells per flask were cultured in T75 flasks containing complete DMEM/F12 media at 37°C, 5% CO₂, and 95% air humidity until they were 80–90% confluent.

Omere C., Richardson L., Saade G.R., Bonney E.A., Kechichian T, & Menon R. (2020). Interleukin (IL)-6: A Friend or Foe of Pregnancy and Parturition? Evidence From Functional Studies in Fetal Membrane Cells. Frontiers in Physiology, 11, 891.

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