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When required, kanamycin (Km) and gentamicin (Gm) were added to final concentrations ranging from 25 to 150 μg/mL.
Afterward, cells were washed once with PBS, and 60 μl of 10% v/v GM were added per well.
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Sephadex G-100 (5 gm) gel was added in 0.1 M phosphate buffered solution (pH 7.0) and allowed to swell overnight and column (1.5 × 65 cm) was packed.
To prepare GM-CSF encapsulated hydrogels, various amounts of GM-CSF were added to mPEG PLGA copolymer aqueous solution and mixed by pipetting on ice.
Fresh IL-4 and GM-CSF were added every other day.
IL-4 and GM-CSF were added to the culture medium every two days for 7 days.
The concentration of most cytokines was generally low, apart from IL-4 and GM-CSF that were added to the cell culture to generate moDC.
For instance, in the 4T1 model, but not in the 4TO7cg model, GM-CSF and TGB were added to the pcDNA3.1-Mage-b vaccine.
-0.375 gm of TBA was added to 75 ml of DW. - 15 gm of trichloroacetic acid (TCA) were added to DW. - 2.1 ml of 11.9 N Hydrochloric acid (HCL) were added to DW. - The solution completed up to 100 ml of DW. -1 ml of kidney homogenate was added to 2 ml of TBA reagent and mixing.
To induce DC maturation, 1000 U ml−1 TNF α (Cellgenix), GM-CSF and IL-4 were added to immature DCs on day 6, and cultures were continued for 3 days.
GM-Fc or control-Fc were added at 10 μg/mL in a final volume of 100 μL of washing buffer (PBS containing 0.5% BSA, 5 mM EDTA and 2 mM NaN3), and incubated for 1 h at 4°C.
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