The experimental mice were sacrificed on the indicated days and their tumors were harvested and rinsed once with prechilled PBS. The tumors were then shredded into pieces and digested in FBS‐free DMEM containing 1 mg/mL collagenase type‐IV and 1 μg/mL deoxyribonuclease I at 37°C for 90 min. After incubation, single tumor cells were filtered through 100‐μm cell strainers (Corning Incorporated) twice to remove any debris and then treated with the RBC lysis buffer for 5 min at room temperature (RT). Next, single tumor cells were blocked with anti‐mouse CD16/CD32 for 10 min at 4°C to block the Fc receptors and then stained with the appropriate fluorescein isothiocyanate‐conjugated antibodies for 30 min at 4°C, followed by once washing with PBS. Intracellular Foxp3+ Treg staining was performed in accordance with the manufacturer's instructions. To prepare single‐cell spleen tissue suspensions, the collected spleens were crushed with tweezers, filtered through cell strainers, and incubated with RBC lysis buffer at RT for 5 min. After washing the cells, they were subjected to analyses by the CytoFLEX Cytometer (Beckman Coulter) equipped with the CytExpert software and then the FlowJo software (TreeStar). The gating strategy is illustrated in Table S2 .
100 μm cell strainer
Manufactured by Corning
Sourced in United States, Japan, Germany
The 100 μm cell strainer is a laboratory equipment designed for cell filtration. It features a polyethylene terephthalate (PET) mesh with a uniform pore size of 100 microns, allowing for the separation and isolation of cells from a heterogeneous sample.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Merck Group (5 mentions)
Collagenase 4, by Merck Group (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Collagenase type 1, by Thermo Fisher Scientific (3 mentions)
Trypsin, by Thermo Fisher Scientific (3 mentions)
Collagenase 4, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Fujifilm (2 mentions)
40 μm cell strainer, by Corning (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Corning (2 mentions)
Dispase, by Thermo Fisher Scientific (2 mentions)
Deoxyribonuclease, by Thermo Fisher Scientific (2 mentions)
Y 27632 dihydrochloride, by Bio-Techne (2 mentions)
Dmem high glucose, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
62 protocols using 100 μm cell strainer
1
Tumor Infiltration and Immune Cell Analysis
2
Tumorsphere Cultivation and Analysis
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Cells were plated into 6-well ultra-low attachment plates (Corning) at a density of 20,000 cells per ml and cultured in CRCSC culture medium. Tumorspheres were cultured for 12 days, collected by centrifugation (600 r.p.m.), fractionated using 100-μm cell strainers (Corning), and counted by a light microscope.
3
Lactobacillus Colonization of Tonsillar Tissue
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Tonsillar tissues were colonized with 15 vaginal Lactobacillus strains (27 blocks per condition) at a starting inoculum of 104 CFU/mL. At day 3 after inoculation with bacteria, all tissue blocks were collected and digested with collagenase IV (5 mg/mL; Gibco BRL) for 30 min with agitation in a Thermomixer at 900 rpm at 37°C. Following digestion, tissue cells were filtered with 100 μm cell strainers (Corning) and washed with 50 mL of PBS. Cells were then suspended in 1 mL of PBS and stained with 1 μl of live/dead Fixable Viability Dye eFluor 450 (EF 450, Invitrogen) for 15 min. After incubation, cells were washed and diluted in staining buffer [PBS, 1% normal mouse serum, 1% normal goat serum, 1 mM ethylenediaminetetraacetic acid (EDTA)] and stained with anti-CD3-allophycocyanin (CD3-APC) for 20 min. After surface staining, cells were permeabilized with the Fix&Perm Cell Fixation and Cell Permeabilization Kit (Invitrogen) then stained for 20 min with anti-Bcl2-PE, a mitochondrial anti-apoptotic antigen. Data were acquired with a Novocyte flow cytometer (ACEA Biosciences, CA, United States) equipped with 405, 488, and 640 nm laser lines using NovoExpress version 1.2.4 software (ACEA Biosciences) and analyzed using the same software.
4
Single-cell Tumor and Spleen Analysis
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Cells grown after drug treatment or incubation with conditioned media were harvested and rinsed once with prechilled PBS. Cells were subsequently stained with an anti-CD16/CD32 antibody for 30 min at room temperature (RT) to block Fc receptors, and were then labeled with the appropriate fluorescein isothiocyanate-conjugated antibodies for 30 min at 4°C, followed by washing once with PBS. To prepare single-cell tumor tissue suspensions for FACS, tumors were digested in FBS-free RPMI-1640 culture medium containing 1 mg/mL collagenase type IV and 1 µg/mL deoxyribonuclease I at 37°C for 90 min and were then filtered through 100 μm cell strainers (Corning Incorporated, Corning, New York, USA). To prepare single-cell spleen tissue suspensions, spleens were crushed with tweezers, filtered through cell strainers, and incubated with RBC lysis buffer at RT for 5 min. The KLN 205-Luc cells were frozen and thawed three times, and the resulting cell debris was used to stimulate the single-cell spleen suspensions overnight. The subsequent steps were the same as described for cell lines. We analyzed the cells with a FACScan cytometer (Becton Dickinson) and acquired 10,000 or 100,000 cells for each sample.
5
Isolation of Vascular Endothelial Cells
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The resected tissue specimens were placed in Dulbecco’s modified Eagle’s medium (DMEM; Fujifilm Wako, Osaka, Japan) supplemented with Antibiotic-Antimycotic (ABAM; Thermo Fisher Scientific, Waltham, MA, United States). The specimens were transported to the laboratory and cut into 1 × 2-cm with full thickness. The VECs were then isolated by washing the tissues three times with DMEM supplemented with ABAM, mincing the tissues into approximately 3-mm3 pieces with scissors, and digesting the mix with Skin Dissociation Kit (Miltenyi Biotech, Bergish Gladbach, Germany) according to the manufacturer’s instructions. Briefly, this process involved placing the tissue mix in a gentleMACS C tube together with DMEM containing 1% bovine serum albumin (BSA), Enzyme P, Enzyme D, and Enzyme A (Miltenyi Biotech). The mixture was then allowed to digest for approximately 1 hour in a GentleMACS Octo Dissociator with Heaters (Miltenyi Biotech). Subsequently, the cells were squeezed through 100-μm Cell Strainers (Corning, Corning, NY, United States) and harvested by centrifugation (300 g, 10 min, 4°C). Thereafter, PEB buffer composed of phosphate-buffered saline (PBS), pH 7.2, 0.5% BSA, and 2 mM EDTA was added to the cells and mixed well.
6
Comprehensive Cell Culture Protocol
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Falcon tubes, culture plates, flasks, and 100 μm cell strainers were obtained from Corning (Corning, NY, USA). Centrifuges included a Rotixa 50 RS (Hettich Zentrifugen, Tuttlingen, Germany). Leica DM750 and Leica DMIL microscope were used for standard light analysis (Wetzlar, Germany). Flow cytometry sample acquisition was performed with an Accuri C6 (Becton Dickinson, Franklin Lakes, NJ, USA) and an Attune NxT (Invitrogen, Carlsbad, CA, USA).
7
Flow Cytometry Analysis of Splenic Immune Cells
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flow cytometry was performed on the spleen samples collected on day 14 after MCAO. The mice were deeply anesthetized and the spleens were removed and collected in a 1.5-ml tube. To obtain single cell suspension, the spleens were grinded in PBS using a mechanical trituration method through a 100-μm cell strainer (Corning, New York, NY). The cell suspension was centrifuged for 5 min at 1000 rpm at 25 °C, and the supernatant was discarded. Red blood cells were removed by re-suspending the cells in red blood cell lysis buffer (NH4Cl 8.29 g, KHCO3 1 g, EDTA-2Na 37.2 mg, diluted in distilled water, at pH = 7.4) for 30 s. Then the cell suspension was centrifuged for 5 min at 1000 rpm at 25 °C again and the supernatant was discarded. After washing in PBS, the cells were re-suspended in 1 ml PBS. Single cell suspension of 200 μl was incubated with fluorescence-conjugated rat–anti-mouse F4/80-FITC (1:200, BioLegend, San Diego, CA) for 30 min at 4 °C. Then the cells were centrifuged for 5 min at 2500 rpm at 25 °C. After discarding the supernatant, the cells were washed once with PBS. Then the cells were re-suspended in 300 μl PBS and analyzed by a flow cytometry (BD Biosciences, San Jose, CA). A minimum of 10,000 events were acquired for each sample.
8
Isolation and Culture of Endometrial Epithelial Cells
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As described in previous studies,14 (link),17 (link) endometrial tissues collected from leiomyoma patients were minced and homogenized immediately, then enzymatically digested in 1 mg/mL of collagenase IV (Sigma, USA) with gentle shaking at 37 °C for 60 min. The cells were filtered through a 100 μm cell strainer (Corning, USA) and then a 40 μm cell strainer (Corning, USA) to remove tissue debris. Glandular elements from the 40 μm membrane were backwashed several times with PBS and transferred to a universal tube and pellet by centrifugation at 300×g for 5 min. The sediment was then collected and resuspended in Dulbecco's modified Eagle's medium (DMEM) (Hyclone, USA) + 10% fetal bovine serum (FBS) (Hyclone, USA) + 1% penicillin/streptomycin (Beyotime Biotechnology, China) and incubated for 14 days at 37 °C and 5% CO2. Antibodies purchased from Abcam plc, USA, were used to identify EECs: rabbit anti-cytokeratin 19 (1:200; ab52625, Abcam plc, USA) and goat anti-rabbit IgG - Alexa Fluor® 488 (1:200; ab150077, Abcam plc, USA).
9
Isolation and Phenotyping of Murine Bone Marrow Cells
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Bone marrow cells were obtained from the femur of female nude mice according to reported procedures 12 , 13 (link). Briefly, cells from bone marrow were pipetted and suspended in RPMI-1640 medium with 10% FBS to give single-cell suspension. Cells were then passed through a 100-μm cell strainer (Corning, New York, NY). For cell counting, the hemocytometer was used. For flow cytometry, fluorescence probes were added, and cells were incubated on ice for 1 h. For each flow cytometry experiment, unstained cell control and single-color compensation controls had been performed. After washing with PBS 3 times, bone marrow cells were examined on a FACSCalibur flow cytometer or LSRFortessa X-20 analyzer (BD Biosciences, San Jose, CA) and analyzed with FlowJo software (Tree Star, Inc., Ashland, OR). About 50,000 bone marrow cells were obtained for each analysis.
10
Mouse Bone Marrow Cell Staining Protocol
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Normal mouse bone marrow cell suspension was obtained from femur and tibia of 8-12 w male Balb/cJ mice. After the bone marrow cells were flushed out with PBS and filtered through 100μm cell strainer (CORNING, Wiesbaden, Germany), the blood corpuscles were removed with the Ammonium-Chloride-Potassium (ACK) buffer (0.15M NH4Cl, 10mM KHCO3, 0.1mM Na2 EDTA, pH 7.2-7.4). Then, one drop of a sample on the glass microscopic slide was spread by using cover glass as a wedge smear technique. The cells were fixed in a 4% formaldehyde solution for 10 minutes at room tempreture and washed with PBS three times. After permeabilization with 0.5% Tween-PBS for 10 minutes at room temperature, they were blocked with blocking buffer (10% normal serum/ 0.3M glycine/ 0.1% BSA, 0.1% Tween-PBS) and stained by the primary antibodies at 4 °C over night. Next day, the slides were washed with PBS three times and stained by the secondary antibody 1000-fold-diluted with blocking buffer containing DAPI (1 µg/ml) for 1 h at room temperature. The smears were viewed, and the images were captured by using a Keyence BZ-X710 all-in-one fluorescence microscope (Osaka, Japan).
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