To make 0.5% agar gels, 2 mL 2x RPMI (made from powder) (Sigma St. Louis, Missouri, USA) was mixed with 200 µl human serum (One Lambda, Los Angles, California, USA) and 800 µl ultrapure H2O. This was prewarmed to 37°C in a water bath. 2% agar was dissolved in ultrapure H2O by bringing it to a boil in the microwave and mixing it on high speed on a vortex mixer for 20 seconds. This process was repeated four times. One mL of the agar solution was added to the prewarmed mixture to make a 0.5% agar medium solution. Of the solution, 800 µl was added to each well of a 4 well 1.5 polymer tissue culture treated chambered coverslip (Ibidi, Martinsried, Germany) that was precoated with 20% human serum in RPMI for 30 min at 37°C. To generate a uniform concentration of CCL19 (PeproTech), CCL19 was added to a final concentration of 100 ng mL-1 before letting the agar solidify. The agar was left to set for 1 hour. Next, a three-pronged bespoke autopsy punch was used to create a line of three wells, each of a three mm diameter and 2 mm apart in the agar (Supplementary Figure 2
1.5 polymer tissue culture treated chambered coverslip
Manufactured by Ibidi
Sourced in Germany
The 1.5 polymer tissue culture treated chambered coverslip is a laboratory equipment designed for cell culture applications. It provides a sterile and optimized environment for the growth and observation of cells.
Automatically generated - may contain errors
2 protocols using 1.5 polymer tissue culture treated chambered coverslip
1
Agar-Based Microenvironment for Chemoattractant Studies
2
Fabrication of 0.5% Agar Gels
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To make 0.5% agar gels, 2 mL 2x RPMI (made from Powder) (Sigma St. Louis, Missouri, USA) was mixed with 200 µl human serum (One Lambda, Los Angles, California, USA) and 800 µl ultrapure H2O. This was prewarmed to 37°C in a water bath. 2% agar was dissolved in ultrapure H2O by bringing it to a boil in the microwave and mixing it on high speed on a vortex mixer for 20 seconds. This process was repeated four times. One mL of the agar solution was added to the prewarmed mixture to make a 0.5% agar medium solution. Of the solution, 800 µl was added to each well of a 4 well 1.5 polymer tissue culture treated chambered coverslip (Ibidi, Martinsried, Germany) that was precoated with 20% human serum in RPMI for 30 min at 37°C. To generate a uniform concentration of CCL19 (PeproTech), CCL19 was added to a final concentration of 100 ng mL -1 before letting the agar solidify. The agar was left to set for 1 hour. Next, a three-pronged bespoke autopsy punch was used to create a line of three wells, each of a three mm diameter and 2 mm apart in the agar (Supplementary Figure 1).
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