Isolated colon tissue was washed three times with HBSS (Gibco) containing 2% FBS, then incubated in 1 mM DTT (Thermo Scientific) in HBSS 2% FBS at 37°C under constant agitation for 15 min. Tissue was then incubated in HBSS 2% FBS 5 mM EDTA (Sigma) for 15 min at 37°C under constant agitation. This process was repeated twice with fresh buffer. Tissue was cut into smaller pieces and incubated with RPMI (Gibco), containing 2% FBS and 3 mg ml−1 collagenase type IV (Sigma) and incubated for 10 min at 37°C under constant agitation. Enzymatic digestion was stopped by adding ice cold RPMI 2% FBS. Cell suspension was first filtered through a 100 μm cell strainer (Greiner) followed by a 40 μm cell strainer (Falcon). Cells were centrifuged at 350 x g for 10 min at 4°C and resuspended in RPMI 2% FBS at a density of 1.5 106 cells ml−1.
Cell strainer
Manufactured by Greiner
Sourced in Germany, Austria
A cell strainer is a laboratory device used to separate cells or other small particles from a liquid suspension. It consists of a fine mesh screen or filter that allows the liquid to pass through while retaining the desired particles.
Automatically generated - may contain errors
Lab products found in correlation
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Merck Group (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Collagenase type 4, by Merck Group (1 mentions)
Cell strainer, by BD (1 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Hanks balanced salt solution (hbss), by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Cd45 apc cy7, by BioLegend (1 mentions)
Ly6g pe cy7, by BioLegend (1 mentions)
Cd34 pe cy7, by BioLegend (1 mentions)
Ly6c pe cy7, by BioLegend (1 mentions)
Cd80 pe cy7, by BioLegend (1 mentions)
Cd86 pe cy7, by BioLegend (1 mentions)
Cd11c pe cy7, by BioLegend (1 mentions)
Sca 1 pe cy7, by BioLegend (1 mentions)
Cd11b pe cy7, by BioLegend (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
17 protocols using cell strainer
1
Isolation of Intestinal Epithelial Cells
2
Comprehensive Flow Cytometric Analysis of Murine Lung Cells
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In brief, lungs were isolated from mice and cut into small pieces. The fragments were digested in DMEM (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, 0.1% collagenase A (Roche)/0.1% dispase (Roche), and 10 U/ml DNase I (Roche) at 37°C for 60 min. The lung fragments were filtrated through a 70‐µm Cell Strainer (Greiner Bio‐One) into phosphate‐buffered saline, containing 5 mM ethylenediaminetetraacetic acid and 0.5% FBS. The single cell suspensions were divided into 1 × 106 cells each per analysis and underwent a flow cytometric analysis with a FACS Versa (BD Biosciences‐Pharmingen). For the staining, fluorescence‐conjugated monoclonal antibodies against the following targets were used at a concentration of 0.25 µg/106 cells in 100 µl volume: CD45 APC/Cy7 or PerCP/Cy5.5, CD11b PECy7 or PerCP/Cy5.5, CD11c PECy7, Gr‐1 PECy7 or APC, CD34 PECy7, CD40 PECy7, CD64 PECy7, CD80 PECy7, CD86 PECy7, IA/IE PECy7, F4/80 PECy7 or APC, CXCR4 biotin, Sca‐1 PECy7, Ly6C PECy7, Ly6G PECy7, MerTK PECy7, all from BioLegend, as were 7‐AAD Viability Staining Solution and PECy7‐conjugated Streptavidin. Each analysis of flow cytometry was performed through three experiments with a total of six mice per group.
3
Tumor Dissociation Protocol for Murine Samples
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Tumor tissues were removed from the skin lesions of the tumor‐bearing mice at the endpoint day and kept in cold FACS buffer (PBS contains 3% FBS). Enzyme mix (Tumor Dissociation Kit, mouse [#130‐096‐730]) was prepared at the time of use. A 2.5‐ml enzyme mix was used for dissociation of tumors that weighed <500 mg, and a 5‐ml mix was used for those that weighed over 500 mg. Tumor samples were cut into 2–3 mm pieces with scissors inside the gentle MACS™ C Tubes (#130‐093‐237) containing the enzyme mix. Dissociation was performed using the gentle MACS Octo Dissociator with Heaters. The 37C_m_TDK_2 program was used for MMTV‐PyMT–derived tumors. A total of 10 ml FACS buffer was added to the dissociated tissues, and the reaction liquid was filtered through a 40‐μm cell strainer (#542040, Greiner Bio‐one). After red blood cell lysis, we counted the numbers of dissociated samples and analyzed them by the flow cytometry analysis (see Section 2.7).
4
Isolation and CFSE Labeling of OT-I CD8+ T Cells
5
Isolation of Nasal Polyp Cells
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All tissue samples were collected intraoperatively from 10 patients undergoing standard paranasal sinus surgery. Additionally, two samples of normal nasal tissue as control group, where collected from two patients undergoing functional septorhinoplasty or uncinectomy because of a maxillary sinus cyst. The polyps and the nasal mucosae were cut into small fragments and mashed through a cell strainer (Greiner Bio-One) from 100 μm to 40 μm in PBS (Gibco). After washing twice in PBS, the cell number and cell viability were determined using a CASY system according to the manufacturer’s protocol. After centrifugation (5 minutes, 1,600 rpm) cells were frozen at –80℃ with 1 mL freezing medium.
6
Cryopreservation of human salivary gland cells
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Human biopsies of non-malignant SMG were minced into small pieces using a sterile disposable scalpel followed by two rounds of mechanically dissociation using the gentleMACS dissociator [Milteny, 130-095-937] and enzymatic digestion in 5 ml HBSS [Gibco, 14175129] with 1% HSA [Sanquin, 15522636] buffer containing 125 µl Collagenase NB6/4 [Nordmark, N0002779], 62.5 µl Pulmozyme [KFF/Roche, RVG 16734] and 625 µl CaCl2 [Pharmacy UMCG, G00115] (per 100 mg tissue). Isolated cells were collected by centrifugation, washed twice with HBSS/1% HSA buffer filtered through a 100 μm cell strainer [Greiner, 542000] and again collected by centrifugation. Pelleted cells were resuspended in Cryostor® CS10 [StemCell Technologies, 07931] with 10 µM Y-27632 at a final concentration of 4–10 million cells per ml, cryopreserved using a Corning® CoolCell [Corning, 432000] which provides freezing at the rate of −1°C/min and stored at −140°C (Figure 1A ).
7
Isolation of Hippocampal Microvessels from Mice
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Microvessels were isolated per previous protocols36 (link),37 (link) with minor modifications.
Briefly, after 4 hours of fasting, mice on either a STD or HFD were euthanized
via cervical dislocation. Pooled hippocampal samples from five mice were
transferred to a 50 mL Falcon tube filled with 2.5–5 ml of 1x PBS containing
protease inhibitor (Roche, Branchburg, NJ). The tissues were minced with a 18,
23, and finally 25-gauge needle/syringe until the media became milky. Cells were
then centrifuged at 4000 × g, 4°C for 10 minutes, and the supernatant was
removed. Pellets were re-suspended in 30% Dextran (31,390, Sigma-Aldrich
Burlington, MA) approximately 25 times at 1:3 pellet:Dextran ratio, and
re-centrifuged at 10,000 × g, 4°C for 10 minutes. The myelin layer and
supernatant were discarded, and the pellet was re-suspended in 50 µl 1x PBS.
Microvessels were then passed through a cell strainer (40 µm, Greiner Bio-one,
Monroe, North Carolina) with 500 µl 1x PBS and treated with 20 nM insulin or
saline for 15 minutes diluted in the 1x PBS, centrifuged at 10,000 × g, 4°C for
5 minutes. The supernate was discarded and the pellet was re-suspended in 300 µl
T-PER with protease inhibitor for analysis. The samples were stored at −80°C
until use for western immunoblotting as described below.
Briefly, after 4 hours of fasting, mice on either a STD or HFD were euthanized
via cervical dislocation. Pooled hippocampal samples from five mice were
transferred to a 50 mL Falcon tube filled with 2.5–5 ml of 1x PBS containing
protease inhibitor (Roche, Branchburg, NJ). The tissues were minced with a 18,
23, and finally 25-gauge needle/syringe until the media became milky. Cells were
then centrifuged at 4000 × g, 4°C for 10 minutes, and the supernatant was
removed. Pellets were re-suspended in 30% Dextran (31,390, Sigma-Aldrich
Burlington, MA) approximately 25 times at 1:3 pellet:Dextran ratio, and
re-centrifuged at 10,000 × g, 4°C for 10 minutes. The myelin layer and
supernatant were discarded, and the pellet was re-suspended in 50 µl 1x PBS.
Microvessels were then passed through a cell strainer (40 µm, Greiner Bio-one,
Monroe, North Carolina) with 500 µl 1x PBS and treated with 20 nM insulin or
saline for 15 minutes diluted in the 1x PBS, centrifuged at 10,000 × g, 4°C for
5 minutes. The supernate was discarded and the pellet was re-suspended in 300 µl
T-PER with protease inhibitor for analysis. The samples were stored at −80°C
until use for western immunoblotting as described below.
8
Lung Cell Colony Formation Assay
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Lungs were removed, minced, and digested with a collagenase IV (Merck Life Science) solution for 140 min 4°C. The suspension was filtered with a cell strainer (Greiner) and centrifuged. Cells were washed and resuspended in DMEM containing thioguanine (10 µg/mL, Merck Life Science) and seeded in 100 mm3 Petri dishes at three dilutions (1:2, 1:10, and 1:100). Colonies were allowed to grow for 2 weeks and then fixed with methanol and stained with crystal violet.
9
Isolation and Characterization of Immune Cells
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For isolation of lymphocytes, spleens were meshed through a metal strainer. Livers were meshed through a cell strainer (Greiner) and lymphocytes purified using a Percoll (Sigma) gradient. Lungs were digested using Collagenase II (Roche, 140 U ml-1) and DNaseI (Sigma,10 µg ml−1) and meshed through a cell strainer. The following mAbs were purchased from BioLegend (BL), eBioscience (eB) or Miltenyi Biotec (MB): anti-CD8α (clone 53.6.7, cat. # 100723 (BL), diluted 1:100), anti-Thy1.1 (HIS51, #14-0900-81 (eB), 1:1000), anti-CD3ε (145-2C11, #100312, 1:100), anti-CD11b (M1/70, #101217 (BL), 1:100), anti-CD27 (LG.7F9, #124211 (BL), 1:500), anti-KLRG1 (2F1, #138418 (BL), 1:150), anti-CD45.1 (A20, #110724 (BL), 1:200), anti-NK1.1 (PK136, #108714, 1:500), anti-CD4 (GK1.5, #47-0041-82 (eB), 1:100), anti-H-2Kb (AF6-885, #116505 (BL), 1:100), anti-H-2Db (KH95, #111508 (BL), 1:50), anti-Qa-1B (6A8.6F10.1A6, #13−105-048 (MB), 1:10). Zombie NIR dye (BioLegend) or DAPI (Sigma) was used for dead cell exclusion. To detect virus-specific CD8+ T cells, lymphocytes were stained with DbGP33 and DbNP396 tetramers57 (produced in-house). Staining was performed for at least 20 min at 4 °C. Samples were measured on a LSR Fortessa or Canto II cytometer (both BD Biosciences) and data were analyzed with FlowJo software 8.8.7 (Tree Star).
10
Isolation and Expansion of Mesenchymal Stem Cells
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Bones were broken into fragments and incubated for 1 h at 37 °C in phosphate-buffered saline (PBS) supplemented with 1 mg/mL collagenase type I (Thermo Fisher, Waltham, MA, USA). Supernatants were filtered in a cell strainer with 100 µm nylon mesh pores (Greiner Bio-One, Kremsmünster, Austria). Afterwards, bone fragments retained in the cell strainer were transferred into StemMACS MSC Expansion Media XF (Miltenyi Biotec, Bergisch Gladbach, Germany) supplemented with 1% penicillin/streptomycin. Then, adherent MSC were expanded in T175 flasks in a humidified 5% CO2 atmosphere at 37 °C and passaged at 80% confluency.
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