70 μm cell strainer

Manufactured by Avantor

Sourced in Denmark, Sweden, Canada, United States

The 70 μm cell strainer is a laboratory filtration device designed to separate and isolate cells from a cell suspension. It features a mesh screen with a pore size of 70 micrometers, which allows the passage of single cells and smaller cellular debris while retaining larger cell aggregates and tissue fragments.

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Lab products found in correlation

Dnase 1, by Merck Group (2 mentions) Penicillin, by Thermo Fisher Scientific (2 mentions) Streptomycin, by Thermo Fisher Scientific (2 mentions) Nylon cell strainer, by BD (1 mentions) Collagenase, by Thermo Fisher Scientific (1 mentions) Dynal mpc s, by Thermo Fisher Scientific (1 mentions) Rat anti mouse cd31, by BD (1 mentions) Agarose, by Merck Group (1 mentions) Collagenase type 2, by Thermo Fisher Scientific (1 mentions) Magnetic activated cell sorting (macs), by Miltenyi Biotec (1 mentions) Hanks balanced salt solution (hbss), by Avantor (1 mentions) Hanks balanced salt solution (hbss), by Merck Group (1 mentions) Collagenase d, by Merck Group (1 mentions) Cytoflex lx flow cytometry, by Beckman Coulter (1 mentions) Red blood cell lysis, by BD (1 mentions) Anti cd19 pe cy7, by BD (1 mentions) Fcs express 6 flow cytometry software, by De Novo Software (1 mentions) Lsr flow cytometer, by BD (1 mentions) Anti cd3e v450 500a2, by BD (1 mentions) Rbc lysis buffer, by BioLegend (1 mentions)

14 protocols using 70 μm cell strainer

Isolation of Endothelial Cells from Liver and Lung

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Livers or lung lobes were collected in dry 10 cm dishes and minced finely with blades for one minute, and then incubated in 25 ml of pre-warmed Dulbecco modified Eagle medium (4.5 g/L glucose with L-glutamine) containing 2 mg/mL collagenase (Invitrogen) in 50 ml tubes, gently shaking for 45 min at 37°C. Suspensions were passed through a 70 μm cell strainer (VWR) and cells were spun down at 400 g for 8 min at 4°C. Pellets were resuspended in 10 ml Dulbecco modified Eagle medium containing 10% FBS, 50 U/ml penicillin and 50 μg/ml streptomycin, passed through 40 μm Nylon cell strainer (BD Falcon, Cat. No. 352340) and centrifuged at 400 g for 8 min at 4°C. Cells were resuspended in cold DPBS (1 ml/lung and 2 ml/liver), added to sheep anti-Rat IgG-coupled Dynabeads (Invitrogen) pre-incubated with purified Rat Anti-Mouse CD31 (BD Pharmingen) and incubated at 4°C for 20 min. The beads were separated using a magnetic particle concentrator (Dynal MPC-S; Invitrogen) and washed with cold DPBS with 0.1% BSA. This washing step was repeated five times after which cells were lysed in RIPA buffer for Western Blotting.

Zhao X., Nedvetsky P., Stanchi F., Vion A.C., Popp O., Zühlke K., Dittmar G., Klussmann E, & Gerhardt H. (2019). Endothelial PKA activity regulates angiogenesis by limiting autophagy through phosphorylation of ATG16L1. eLife, 8, e46380.

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Establishment of Patient-Derived Tumoroids

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Patient-derived tumoroids were established as described by Jeppesen et al. [39 (link)] with minor modifications. In short, freshly resected tumour samples of 0.01–1 g were divided into 1 mm³ pieces and digested for 20 min at 37⁰C with 1 mg/ml collagenase type II (Thermo Fisher Scientific) diluted in STEM medium. Subsequently, the tissue suspension was sequentially filtered through a 70 μm cell strainer (VWR, Soeborg, Denmark) and a 30 μm pre-separation filter (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany). Tissue retained by the 70 μm filter was collected and redigested for 10 min at 37⁰C and passed through the filters again. This step was repeated until all tissue could pass through the 70 μm filter. Retained tumour fragments (30–70 μm) were seeded in STEM cell medium in petri dishes coated with 1.5% agarose (Sigma-Aldrich) and cultured at 37⁰C in a 5% CO₂ humidified incubator. After 3–5 days the tissue fractions which had densified into rounded tumoroids with smooth surfaces could be collected for further culture. Tumoroids established from colonic tumours were denoted C after the patient number and tumoroids from liver metastases were denoted L.

Truelsen S.L., Mousavi N., Wei H., Harvey L., Stausholm R., Spillum E., Hagel G., Qvortrup K., Thastrup O., Harling H., Mellor H, & Thastrup J. (2021). The cancer angiogenesis co-culture assay: In vitro quantification of the angiogenic potential of tumoroids. PLoS ONE, 16(7), e0253258.

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Isolation of Testicular Cells from Animal Tissue

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Fresh testicular tissues recovered from castrated animals were transported to the lab at 4 °C in HBSS (Sigma-Aldrich, H9269) containing 10 units/mL of penicillin and 10 μg/mL of streptomycin (Thermofisher Scientific, Ghent, Belgium, 15140122) and dissected in small fragments. Tissues from four animals were digested in HBSS supplemented with 1 mg/mL of collagenase D (Sigma-Aldrich, 11088858001) and 1 mg/mL of DNase I (Sigma-Aldrich, 11284932001) for 20 min at 37 °C with repeated flushing every 3–4 min. Cellular suspensions were passed through a 70-μm cell strainer (VWR, 732-2758) and rinsed two times with HBSS to eliminate residual collagenase and DNase. Following counting with trypan blue and a Bürker chamber, testicular cells were used for preparation of TOs.

Vermeulen M., Del Vento F., Kanbar M., Pyr dit Ruys S., Vertommen D., Poels J, & Wyns C. (2019). Generation of Organized Porcine Testicular Organoids in Solubilized Hydrogels from Decellularized Extracellular Matrix. International Journal of Molecular Sciences, 20(21), 5476.

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Multiparameter Flow Cytometry Analysis

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Spleen and lymph node samples were harvested as described above. The samples were sliced, meshed, and passed through a 70 μm cell strainer (VWR International, Sweden) to generate single-cell suspensions. Single cells were stained for the Treg panel and T-cell panel (Supplementary Table S1) and were analyzed in the CytoFLEX LX flow cytometry (Beckman Coulter Life Sciences). Single cells from lymph nodes were also stained for mouse lymph node peripheral T_RM cells and DC panel (Supplementary Table S1) and evaluated using BD melody (BD Biosciences) for tissue-resident memory CD8⁺ T cells. The CD11c⁺ cells were also sorted for further analysis. The blood was collected as described above and treated with red-blood-cell lysis (BD Biosciences). The cells were stained by a tumor-matching markers panel (Supplementary Table S1) and the stained cells were assessed in CytoFLEX LX flow cytometry (Beckman Coulter Life Sciences). The data were analyzed and visualized by Flowjo (BD Biosciences).

Jin C., Ali A., Iskantar A., Fotaki G., Wang H., Essand M., Karlsson-Parra A, & Yu D. (2022). Intratumoral administration of pro-inflammatory allogeneic dendritic cells improved the anti-tumor response of systemic anti-CTLA-4 treatment via unleashing a T cell-dependent response. Oncoimmunology, 11(1), 2099642.

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Immune Cell Isolation from Murine MLN

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Mechanical digestion of the MLN was achieved by a syringe plunger. After mechanical digestion, single cells were obtained by passing the cells through a 70-μm cell strainer (VWR International, Radnor, PA). Red blood cells (RBC) were lysed using RBC lysis buffer (BioLegend, San Diego, CA). Cells from 2 mice in the same experimental groups were combined to increase cell numbers for analyses. For T-cell populations, cells (1 × 10⁶ cells) were stained with antibodies anti–CD3e-V450 (500A2; BD Biosciences, Franklin Lakes, NJ) and anti–CD4-PE-Cy5 (RM4-5; BD Biosciences). For B-cell populations, cells (1 × 10⁶ cells) were labeled with anti–CD19-PE-Cy7 (1D3; BD Biosciences) and anti–MHC-II-FITC (2G9; BD Biosciences). Populations of T and B cells then were determined by a BD LSR flow cytometer (BD Biosciences ). Flow cytometry data were analyzed using FCS Express 6 Flow Cytometry Software (De Novo Software, Glendale, CA). The gating strategy used to determine immune cell populations is presented in Supplemental Figure 1.

Lin C.Y., Lee A.H., Chiu K.K., Vieson M.D., Steelman A.J, & Swanson K.S. (2020). Saccharomyces cerevisiae Fermentation Product Did Not Attenuate Clinical Signs, but Psyllium Husk Has Protective Effects in a Murine Dextran Sulfate Sodium–Induced Colitis Model. Current Developments in Nutrition, 4(11), nzaa159.

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Isolation and Culture of Immune Cells

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At day 9 after infection, mice were sacrificed and blood were collected for serum. The spleens and livers were collected and processed into single-cell suspensions. Red blood cells in the spleen cells were routinely lysed with ACK lysis buffer, and the cells were washed with PBS and resuspended in in complete tissue culture medium (DMEM supplemented with 10% heat-inactivated fetal bovine serum, 2 mmol L-glutamine, 100 U/mL Penicillin, and 100 μg/ml streptomycin). The liver samples were perfused through the right ventricle with 10 ml of ice-cold-PBS and digested with collagenase-D (125 μg/mL) for 30 min at 37°C before being homogenized. The cells were passed through a 70 μm cell strainer (VWR, Mississauga, ON, Canada) and washed by spinning at 1,200 rpm for 5 min. Washed liver cells were resuspended in 40% percoll (Sigma) and carefully layered above 70% percoll and centrifuged at 750 × g for 20 min at 22°C. The interface containing lymphocytes was collected and washed with PBS. The lymphocytes were resuspended at final concentration of 4 × 10⁶/ml in complete tissue culture medium and cultured for 48 h. The culture supernatant fluids were collected and assayed for cytokines by ELISA.

Kuriakose S., Onyilagha C., Singh R., Olayinka-Adefemi F., Jia P, & Uzonna J.E. (2019). TLR-2 and MyD88-Dependent Activation of MAPK and STAT Proteins Regulates Proinflammatory Cytokine Response and Immunity to Experimental Trypanosoma congolense Infection. Frontiers in Immunology, 10, 2673.

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Isolation and Culture of Adult DPCs

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Adult (30–80 years old) human DPCs were isolated from thigh or scalp tissue according to previously published methods (Hagner and Biernaskie, 2013 ). Tissue was cleaned of underlying fat, minced, and incubated in Dispase (5 mg/mL) at 37°C for 3–4 h. Tissue was washed in Hank’s balanced salt solution (HBSS) and the epidermis removed from the dermis using fine forceps. Remaining tissue was minced and then incubated in collagenase type XI (2×, Sigma) and DNase I (1×, Sigma) for 3–4 h, after which 1% fetal bovine serum (BioWhittaker) was added to inhibit enzymatic activity. Cells were triturated, diluted in HBSS and filtered through a 70 μm cell strainer (VWR) then plated in DMEM:F12 (3:1, Invitrogen) containing 1% (v/v) penicillin/streptomycin (BioWhittaker), 1 μg/mL Fungizone (Invitrogen), 2% (v/v) B27 supplement (Invitrogen), 40 ng/mL fibroblast growth factor 2 (FGF2) (10 μg/mL, BD Biosciences), and 25 ng/mL PDGF-BB (25 μg/mL, R&D Systems); hereafter referred to as Proliferation Medium (PM) (1×). Primary cultures were prophylactically treated with Plasmocin (0.02% [v/v], Invitrogen) and absence of Mycoplasma in all cultures was confirmed by PCR.

Agabalyan N.A., Sparks H.D., Tarraf S., Rosin N.L., Anker K., Yoon G., Burnett L.N., Nickerson D., Di Martino E.S., Gabriel V.A, & Biernaskie J. (2019). Adult Human Dermal Progenitor Cell Transplantation Modulates the Functional Outcome of Split-Thickness Skin Xenografts. Stem Cell Reports, 13(6), 1068-1082.

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Placenta Cell Isolation and Analysis

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The decidua was dissected away from the placentas, which were then washed in ice-cold PBS and cut into small fragments. The fragments were transferred to 1.5 mL digestion medium (DMEM with 500 U/mL Collagenase Type I and 2 U/mL DNAse I) and passed through an 18G syringe needle several times to further dissociate cells. The digestion mixture was incubated for 35 min at 37°C with 1000 RPM shaking using a VWR Shake-Touch block heater. After this incubation period, the mixture was filtered through a 70 μm cell strainer (VWR) and rinsed with an equivalent volume of cold PBS with 10% FBS. The tissue samples were centrifuged at 5,000×g for 5 min at 4°C, and the supernatant was removed. Then the sample was incubated with red blood cell lysis buffer at room temperature for 5 min. The sample was centrifuged as above, and the supernatant was discarded. The cells were then washed with 10% FBS in PBS a final time. The cells were divided into equal samples for fixation and staining. Briefly, cells were incubated with LIVE/DEAD^™ Fixable Yellow Dead Cell Staining Kit according to manufacturer’s instructions (ThermoFisher). Cells were then fixed using Flow Cytometry Fixation Buffer for 20 min on ice. Cells were resuspended in 10% FBS in PBS and analyzed using a NovoCyte 3000 (ACEA Biosciences). Data was analyzed using NovoExpress.

Mechetner E.B., Schott B., Morse B.S., Stein W.D., Druley T., Davis K.A., Tsuruo T, & Roninson I.B. (1997). P-glycoprotein function involves conformational transitions detectable by differential immunoreactivity. Proceedings of the National Academy of Sciences of the United States of America, 94(24), 12908-12913.

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Epidermal Cell Isolation and Sorting

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The dorsal skin was dissected and laid flat for subcutaneous fat
scratching and then floated epidermis side up in 0.25 % Trypsin (Gibco) in
PBS for 1 hour at 37°C to separate epidermal and dermal layers.
Following the incubation, the epidermis was carefully scraped off and
transferred to 10% FBS (fetal bovine serum) in DMEM (Gibco), in which it was
manually gently dissociated using a serological pipette. The entire volume
was filtered through a 70 μm cell strainer (VWR) to obtain a single
cell suspension that was then centrifuged and resuspended in 2 % FBS in PBS.
For cell staining, antibodies were directly added to the cell suspension and
incubated for 10 min on ice. Afterwards, cells were washed using 2 % FBS PBS
and resuspended for sorting in 5 mM EDTA PBS. The following antibodies were
used: anti-CD49f-APC (1:300, 313615, BioLegend), anti-CD49f-PercP/Cy5.5
(1:300, 313618, BioLegend), anti-CD49f-FITC (1:300, 313606, BioLegend),
anti-CD34-PE (1:50, 551387, BD Pharmingen), anti-CD34-Alexa Fluor 647 (1:50,
560230, BD Pharmingen), anti-Sca1-Ly-6A/E-Violet 605 (1:200, 108133,
BioLegend) and DAPI (1:1000, Biotium). Flow cytometry was performed on a BD
LSRII cytometer (BD Biosciences), sortings on a BD FACS Aria II sorter (BD
Biosciences). Flow cytometry data was collected and exported using BD FACs
Diva software (BD Biosciences) and analyzed and plotted using FlowJo
software.

Huang S., Kuri P., Aubert Y., Brewster M., Li N., Farrelly O., Rice G., Bae H., Prouty S., Dentchev T., Luo W., Capell B.C, & Rompolas P. (2021). Lgr6 marks epidermal stem cells with a nerve-dependent role in wound re-epithelialization. Cell stem cell, 28(9), 1582-1596.e6.

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Isolation of Intestinal Cells from Tadpoles and Adults

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After euthanasia, whole tadpole and adult intestines were excised from animals using sterilized dissection tools. Tissues taken for histology were processed as described below. For cell isolation, whole intestines were incubated in Liberase (0.1mg/ml, Roche Diagnostics) diluted with A-PBS for 30 minutes at 27°C and then washed 1x with A-PBS. Cells used for staining were further passed through a 70 μm cell strainer (VWR, Radnor, Pennsylvania, USA) to produce single-cell suspensions. The viability of the isolated intestinal cell was confirmed by Trypan blue exclusion.

Hauser K.A., Singer J.C., Hossainey M.R., Moore T.E., Wendel E.S., Yaparla A., Kalia N, & Grayfer L. (2021). Amphibian (Xenopus laevis) Tadpoles and Adult Frogs Differ in Their Antiviral Responses to Intestinal Frog Virus 3 Infections. Frontiers in Immunology, 12, 737403.

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