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A slightly changed version of this medium was used throughout most experiments for previous studies (B).
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Most experiments run for months, or perhaps a year or two.
In most experiments, cells for RNA extraction were collected from 60.1-cm Petri dishes.
Cells seeded into 6-well plates or 9-cm dishes were transfected twice in most experiments (except for the knockdown of PLK1 and SGOL1), with a 24 hour time-interval, using 40 nM siRNA to obtain efficient depletion.
2 x Yan-YFP animals used for most experiments were null for endogenous yan and carried a single copy of the His2Av-mRFP transgene (yan ER443/yan E884; Yan-YFP/Yan-YFP, His2Av-mRFP).
Cells were grown to mid-log (OD 0.4 0.6) at 30°C for most experiments or at 25°C for experiments involving trf4Δ mutants.
For most experiments, rats were handled for 2 days, given a subcutaneous (SC) needle stick on day 3 and then injected with sesame oil (SC) for 2 days (days 4 and 5).
We used 100 ms integration time for both GFP and mCherry in most experiments (200 ms for competitive recruitment of Nuf2-GFP/Nuf2-mCherry).
For most experiments, subject mice lived together for 2 4 weeks prior to conditioning.
For most experiments, 1000 frames were collected for a total acquisition time of approximately 3.5 seconds.
The medium was removed and replaced with RPMI 1640 containing 5% fetal bovine serum (FBS) for most experiments, or Opti-MEM (Sigma) for experiments designed to measure HMGB1 in conditioned media.
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