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Afterwards, cells were washed once with 1 ml of PBS, and 1 ml of 10% v/v GM was added per well.
To identify the differentially regulated genes when GM was added after washing relative to the inhibitory condition (proliferation effect), and to then perform reverse causal reasoning analysis (see details below), pairwise comparisons were computed with linear models for microarray data (limma) following a global linear model.
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When required, kanamycin (Km) and gentamicin (Gm) were added to final concentrations ranging from 25 to 150 μg/mL.
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.
After that, culture was spitted for control (no Gm) and Gm-variant that was added by Gm to a desirable concentration.
To determine whether this increased number of migrated cells was due to the reduction of GM-CSF concentration in RNF13-KO conditioned medium, recombinant GM-CSF was added to RNF13-KO conditioned media, which led to a reduction in the number of migrated cells (Fig. 5B and 5D).
To test whether GM-CSF could overcome the failure to develop CD103+ DCs in Batf3−/− Flt3L-stimulated cultures, GM-CSF was added to DC cultures 2 days before analysis.
Culture was continued for 9 days and fresh culture medium with GM-CSF was added on days 3, 6 and 8. On day 9, the supernatant containing the dendritic cells was removed and RPMI containing FBS and antibiotics was added.
Fresh medium containing GM-CSF was added every second day.
One hour later, fresh MDDC medium containing IL-4 and GM-CSF was added.
On day 3, 10 ml of complete medium containing 15 ng/ml GM-CSF was added.
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