Tumor spheres were generated by culturing suspended tumor cells from monolayer culture in serum free medium (SFM) containing DMEM/F12 (Gibco), 20 ng/ml EGF, 10 ng/ml bFGF, 10 ng/ml IGF and 2% B27 supplement (Gibco) in ultra-low attachment 24-well plate (Corning) at 37°C/5% CO2. For sphere propagate assay, the first generation spheres were trypsinized and sieved through a 40-μm nylon mesh cell strainer (Fisher Scientific) to obtain a single cell population. 200 cells per well were resuspended in 500 μl SFM, seeded in ultra-low attachment 24-well plate (Corning) at 37°C/5% CO2 for 24 hours, and treated with indicated antibodies for 2 weeks. Cells were fed with 100 μl SFM every 3 – 4 days. Each well was sectionally scanned using the BIOREVO digital microscope (BZ-9000; Keyence) and merged to display the whole well image. Spheres > 100 μm in diameter were counted.
40 μm nylon mesh cell strainer
Manufactured by Thermo Fisher Scientific
The 40-μm nylon mesh cell strainer is a laboratory equipment used to filter and separate cells or other particulate matter. It features a nylon mesh with a 40-micron pore size that allows the passage of single cells or small particles while retaining larger aggregates or debris.
Automatically generated - may contain errors
3 protocols using 40 μm nylon mesh cell strainer
1
Tumor Sphere Formation and Propagation Assay
2
FACS Analysis and Cell Sorting Protocol
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Example 5
FACS Analysis and Cell Sorting
Briefly, cells were completely dissociated in Versene (0.04% EDTA), washed once with phosphate-buffered saline (PBS) by centrifugation at 300 g for 5 min, then resuspended in 0.5 mL Opti-MEM, passed through a 40 μm nylon mesh cell strainer (Fisher Scientific) into a 5 mL round-bottom tube (BD Biosciences) and placed on ice. Cell sorting and analysis was carried out using a FACS Aria II flow cytometer (BD Biosciences) and BD FACSDiva software. The sort gate was a combination of the wild-type live cell gate (from FSC-H vs. SSC-H dot plot) and the events in the top 25% fluorescence (from the FITC histogram) determined for 10,000 events using a 488 nm wavelength. Fluorescent cells were collected in Opti-MEM and expanded in DMEM, 10% FBS, Pen/Strep, 1 μg/mL blasticidin before preparing frozen stocks.
3
Quantifying Blastomyces Lung Burden
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Blastomyces cells were harvested from HMM plates with PBS, passed through a 40-μm nylon mesh cell strainer (Fisher Scientific), and counted on a hemocytometer. Cells were resuspended in PBS at a density of 106/ml, and 20,000 cells (20 μl) were injected intratracheally into male C57BL6/NCr mice (National Cancer Institute via Charles Rivers Laboratories). Lungs were harvested at 10 to 15 days postinfection and placed into 2 ml of PBS for homogenization with a motorized tissue grinder (Omni International, Kennesaw, GA). Lung homogenates were diluted by a factor of 20 to 2 × 105 and plated onto BBL brain heart infusion (Becton, Dickinson Difco, Sparks, MD) agar plates. Colonies were counted 4 to 8 days later, and the total numbers of CFU per lung were displayed as box-and-whisker plots. In each experiment, 10 mice per strain were injected; typically, several mice in an experiment died from complications of the surgery or succumbed to Blastomyces infection prior to euthanasia. In no case did the sample size fall below 6 mice per strain, and the majority of strains (90%) had 8 to 10 mice per group per experiment.
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