Cells from spleen and peripheral blood were isolated as described (24 (link)). Spleens were passed through a 70-µm cell strainer (BD Falcon), and red blood cells were lysed using ACK lysing buffer (Gibco Laboratories). Peripheral blood from the retro-orbital sinus was underlaid with Histopaque-1077 (Sigma-Aldrich). The gradient was centrifuged at 2000 rpm without braking for 20 min at 20°C, and mononuclear cells were recovered from the interface. To isolate tumor-infiltrating lymphocytes, tumors were removed, weighed, and chopped into small pieces. Following incubation in 1 mg/ml collagenase IV (Worthington Biochemical Corp.) and 0.01 mg/ml DNase (Sigma-Aldrich) in RPMI containing 10% FBS and 1% penicillin/streptomycin at 37°C for 30 min, digested cells were passed through a 70-µm cell strainer (BD Falcon), and red blood cells were lysed using ACK lysing buffer (Gibco Laboratories). After washing with RPMI containing 2% FBS and 1% penicillin/streptomycin, cells were counted using a hemocytometer.
70 m cell strainer
Manufactured by BD
Sourced in United States, United Kingdom, Germany, Italy
The 70 μm cell strainer is a laboratory equipment designed for the filtration and separation of cells. It features a nylon mesh with a pore size of 70 micrometers, which allows for the effective removal of cell aggregates and debris from cell suspensions.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Merck Group (17 mentions)
Dnase 1, by Roche (16 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (14 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (11 mentions)
Collagenase d, by Roche (9 mentions)
Trypsin edta, by Thermo Fisher Scientific (8 mentions)
Lsrii flow cytometer, by BD (7 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (7 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (6 mentions)
Collagenase 4, by Merck Group (6 mentions)
Hyaluronidase, by Merck Group (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (5 mentions)
Red blood cell lysis buffer, by Merck Group (5 mentions)
Dmem f12, by Thermo Fisher Scientific (5 mentions)
Collagenase type 4, by Merck Group (5 mentions)
Facsaria, by BD (5 mentions)
Lsrfortessa, by BD (5 mentions)
Fetal bovine serum (fbs), by Merck Group (5 mentions)
Dnase, by Merck Group (5 mentions)
306 protocols using 70 m cell strainer
1
Isolation of Immune Cells from Spleen, Blood, and Tumors
2
Isolation and Culture of Prostate Tumor Cells
3
Isolation and Differentiation of Murine Dendritic Cells
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Mice were anesthetized by intraperitoneal injection of 0.1 ml 2% sodium pentobarbital (Sigma, Saint Louis, Missouri, USA) solution and then euthanized with cervical dislocation. The collection of DCs was performed as previously described [15 (link)]. The cell suspensions in complete medium were filtrated with 70-µm cell strainer (BD Bioscience, Franklin Lakes, New Jersey, USA) and erythrocytes were lysed with lysing buffer (BD Bioscience). Cells were suspended in complete medium with IL-4 (10 ng/ml, R&D Systems, Tustin, California, USA) and granulocyte-monocyte colony-stimulating factor (GM-CSF, 10 ng/ml, R&D Systems). At day 7, lipopolysaccharide (1 µg/ml, Sigma) was added and at day 8 the suspending mature DCs were collected for further research. In addition, following the GentleMACS protocol of the digestion of the spleens, single-cell suspension was obtained with GentleMACS Dissociator (Miltenyi, Auburn, Washington, USA) and then filtrated with 70 µm cell strainer (BD Bioscience). Erythrocytes were lysed with lysing buffer (BD Bioscience). CD3+ T lymphocytes were isolated and purified with Mouse T Lymphocyte Enrichment Set (BD Bioscience).
4
Immune Cell Profiling of Spinal Cord Lesions
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For analysis of immune cell populations around the lesion site, mice were sacrificed, spinal cord from approximately T6–L2 isolated and transferred to ice-cold PBS (Sigma-Aldrich). Tissue was cut in small pieces and digested in RPMI containing 2% fetal calf serum (Sigma-Aldrich), 25 mm HEPES (Sigma-Aldrich), DNase I (10 ng/ml, StemCell Technologies) and Collagenase D (0.8 mg/ml, Roche) for 30′ at 37 °C. Reaction was stopped by adding 1:100 dilution of a 0.5 M EDTA (Sigma-Aldrich) solution. Suspension was filtered through 70 µm cell strainers (Falcon) and re-suspended in a 30% solution of Percoll (Sigma-Aldrich). After 30′ of gradient centrifugation at 10.800 r.p.m., the top (myelin) and lower (red cells) layers were removed and the remaining solution was filtered through 70 µm cell strainers (Falcon). Stainings were performed in ice-cold PBS after Fc-receptor blockade (CD16/32, 1 µl/million cells, clone 2.4G2, BD Biosciences) using LIVE/DEAD staining (1:100, Invitrogen) and the following antibodies: CD45 (1:400, clone 30-F11, BioLegend), CD11b (1:300, clone M1/70, BD Biosciences), Ly6C (1:200, clone AL21, BD Biosciences), Ly6G (1:200, clone 1A8, Biolegend), CD4 (1:200, clone RM4-5, BioLegend). Samples were acquired on a LSR-Fortessa cytometer (BD Biosciences) and results analyzed by FlowJo software.
5
Isolation and Culture of Primary Mouse Hepatic Stellate Cells
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Immortalized human semi-activated HSCs and LX-2 cells were kindly donated by Dr. S. L. Friedmann (Mount Sinai School of Medicine, NY, USA), and maintained in Dulbecco’s modified Eagle’s medium (DMEM), containing 10% fetal bovine serum, 50 units/mL penicillin/streptomycin at 37 °C in a humidified 5% CO2 atmosphere. Primary HSCs were isolated from the livers of 8-week-old ICR mice (Oriental Bio, Sungnam, Korea) as previously reported [11 (link),28 (link)]. After intubation through the portal vein, the livers were perfused in situ with Ca2+-free Hank’s balanced saline solution at 37 °C for 15 min and then perfused with a solution containing 0.05% collagenase and Ca2+ for 15 min at a flow rate of 10 mL/min. The perfused livers were minced, filtered through a 70 m cell strainer (BD Biosciences), and centrifuged at 50× g for 3 min to separate the supernatant and pellet. The pellet was then discarded. Next, the supernatant was further centrifuged at 500× g for 10 min, resuspended in Ficoll plus Percoll (1:10; GE Healthcare, Chicago, IL, USA), and centrifuged at 1400× g for 17 min. HSCs were collected from the interface.
6
Isolation and Culture of Muscle-Derived Cells
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The tissue samples were washed with normal saline to remove blood cells and transferred to sterile Dulbecco's modified Eagle's medium (DMEM; Wako Pure Chemicals Industries, Osaka, Japan) supplemented with 1% penicillin-streptomycin.
The tissue was minced and digested with 0.2% collagenase (Wako Pure Chemicals Industries) and 0.1% DNase I (Sigma-Aldrich, St. Louis, MO) for 1 h at 37 °C. PBS was added to the digested muscle tissue samples, which were filtered through a 70-m cell strainer (BD Biosciences, Franklin Lakes, NJ) and centrifuged at 700 x g for 20 min. The pellets were resuspended in 1 ml staining solution composed of 1% bovine serum albumin (BSA; Sigma-Aldrich) in PBS and then incubated with an Fc receptor blocking solution (Human TruStain FcX, 1:20 in staining buffer; Biolegend, San Diego, CA) for 10 min. The cells were seeded on 24-well chamber slides coated with Matrigel
The tissue was minced and digested with 0.2% collagenase (Wako Pure Chemicals Industries) and 0.1% DNase I (Sigma-Aldrich, St. Louis, MO) for 1 h at 37 °C. PBS was added to the digested muscle tissue samples, which were filtered through a 70-m cell strainer (BD Biosciences, Franklin Lakes, NJ) and centrifuged at 700 x g for 20 min. The pellets were resuspended in 1 ml staining solution composed of 1% bovine serum albumin (BSA; Sigma-Aldrich) in PBS and then incubated with an Fc receptor blocking solution (Human TruStain FcX, 1:20 in staining buffer; Biolegend, San Diego, CA) for 10 min. The cells were seeded on 24-well chamber slides coated with Matrigel
7
Isolation and Purification of Lymphocytes and Splenocytes
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Lymphocytes from anticoagulated peripheral blood were isolated using Histopaque-1077 (Sigma-Aldrich). Splenocytes were isolated by mechanical disruption, followed by digestion with collagenase D (0.4 U/ml; Roche) for 30 min at 37°C with gentle shaking. EDTA was added to a final concentration of 5 mM, and spleens were homogenized by passing through a 70-m cell strainer (BD Biosciences). Red blood cells were lysed using ACK Lysing buffer (Lonza). Cells were washed and resuspended in RPMI 1640 supplemented with 5% fetal bovine serum (FBS).
8
In Vivo Tumor Immune Cell Isolation
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For in vivo experiments, the tumors were weighed and digested at 37°C in 10 mL digestion solution (PBS supplemented with type I collagenase (200 U/mL), hyaluronidase and DNase I (100 µg/mL)) for 1 hour. Single cell suspensions were obtained by grinding the digested tissues and filtering through a 70 µm cell strainer (Becton and Dickinson, BD, USA). The immune cells were freshly isolated using density gradient centrifugation and stained with antibodies for 30 min at 4°C. The following monoclonal anti-mouse antibodies were used: CD45-PECy5.5 (eBioscience, USA), CD3-PECy7 (eBioscience, USA), CD4-APC (BioLegend, USA), CD8-APC (eBioscience, USA), F4/80-APC (Sungene, China), CD11b-PECy7 (eBioscience, USA) and MHC II-PE (eBioscience, USA). Flow cytometry was performed and the data were analyzed using FlowJo software (TreeStar, Ashland, Oregon, USA).
9
Isolation and Culture of Mononuclear Cells
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BMAs were mixed with PBS (Gibco Invitrogen, Grand Island, NY, USA) at a 1:1 ratio. The mixture was loaded onto a histopaque layer (1.077 g/mL; Sigma chemical co., St. Louis, MO, USA). MNCs were separated by density gradient centrifugation (400 ×g, 25 min, room temperature), washed thrice with alpha minimum essential medium (αMEM; Gibco Invitrogen, Grand Island, NY, USA), filtered using a 70-µm cell strainer (Becton Dickinson, Falcon, Germany), and resuspended. The cells were incubated at 37 °C with 5% CO2 in basic medium (αMEM containing 10% FBS, 100 units/mL penicillin, 100 µg/mL streptomycin). The culture flask was washed with PBS to remove the non-adherent cells and incubated further until adherent cells reached confluence. The confluent cells were trypsinized (0.25% trypsin EDTA), divided into several culture flasks, and incubated in the basic medium. This subculture was performed for further studies, and the same procedure was repeated for HVB.
10
Isolation of Bone Marrow Nucleated Cells
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