To address chemokine consumption, BMDMs were cultured in prewarmed RPMI-1640 medium (Hyclone, SH30027.01) containing either 0, 10, or 100 ng/ml CCL3 or CCL2. BMDMs were incubated at 37°C, 5% CO2 for 18 h and the supernatants subsequently removed and analyzed by ELISA (Thermofisher, Ready-Set-Go). For chemokine receptor downmodulation and recycling, thioglycolate-elicited peritoneal macrophages from WT and VISTA KO mice were seeded in prewarmed RPMI-1640 medium (Hyclone), supplemented with 10% FBS and 2 mM HEPES at a density of 2 million cells/ml in round-bottom polypropylene tubes (Corning) containing either 100 ng/ml CCL3, 100 ng/ml CCL2, or 500 ng/ml CCL5. Cells were incubated for the following times: 0, 2, 4, and 7 h. All recombinant chemokines were purchased from Peprotech. To investigate chemokine-induced recycling, cells were treated as described above for 2 h at 37°C, then were placed on ice and immediately washed twice in RPMI medium to remove free chemokines. Cells were then incubated at 37°C for 1 h to allow for receptor recycling and stained with antibodies directed against the various chemokine receptors assayed as described in flow cytometry and analysis.
Round bottom polypropylene tubes
Manufactured by Corning
Round-bottom polypropylene tubes are laboratory equipment designed for various scientific applications. These tubes are made of polypropylene, a durable and chemically resistant plastic material. The round-bottom shape provides stability and allows for efficient mixing and centrifugation. The tubes are available in different sizes and volumes to accommodate different sample volumes and experimental requirements.
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Lab products found in correlation
2 protocols using round bottom polypropylene tubes
1
Chemokine Consumption and Receptor Dynamics
2
MSC Loading into Gelatin Nanovials
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MSCs were loaded into nanovials by pipet-mixing nanovials and the cell solution in 5 ml round-bottom polypropylene tubes (Corning Falcon), which provide a cell-adhesion resistant material and vent cap for gas exchange during incubation. First, the 5x diluted nanovial suspension was reconstituted in complete MSC media. MSCs were detached from tissue culture flasks using TrypLE (Gibco) and resuspended in media at concentrations of 0.9, 1.5 and 2.2 million per ml. Cells and nanovials are then pipette-mixed 10 times in a 3:1 volume ratio for final cell to nanovial ratios of 0.4:1, 0.7:1, and 1:1, respectively. A total volume of less than 0.5 ml per tube was maintained throughout to reduce cell clumping during incubation. The nanovial suspensions were incubated at 37°C and 5% CO2 for 2 hours to allow MSCs to bind to the gelatin coating in nanovial cavities. Then, nanovial suspensions were first strained through a 20 µm strainer (CellTrics) to remove unbound cells. Subsequently, recovered nanovials were strained through a 37 µm strainer (STEMCELL) to remove any large cell/nanovial aggregates. The 1:1 cell to nanovial ratio was used for remaining experiments.
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