Rabbit anti-LAMP1 monoclonal antibody (9091, immunofluorescence 1:100) was from Cell Signaling. Anti-rabbit IgG (H+L) CF350 (SAB4600412, immunofluorescence 1:200), Flag (M2, immunofluorescence 1:1,000 F7425, western blot 1:3,000), Flag M2 agarose (immunoprecipitation 10 μl beads per reaction) were from Sigma. The PI4KB antibody (611816, western blot 1:1,000, immunofluorescence 1:200) and GM130 antibody (610822, immunofluorescence 1:1,000) were from BD Biosciences. For cell treatment that requires medium change, fresh media were pre-warmed overnight in an empty dish in the same incubator, which minimizes disturbance to cells caused by medium change. Chemicals, silica, or tau fibrils were directly added to cell culture media to induce lysosomal membrane damage with equal volume of vehicle (ethanol or DMSO) added into control wells. Tau fibrils (SPR-329) were from StressMarq. Nano-silica (Invivogen, tlrl-sio-2) was suspended in water and sonicated right before each use. Gly-Phe-β-naphthylamide (GPN, Cayman Chemical, 14634), Brefeldin A (Sigma, B5936), ML-SA1 (Cayman Chemical, 29958), MEK6–83 (Cayman Chemical, 21944), O-methyl-serine dodecylamide hydrochloride (MSDH, Avanti Polar Lipids, 850546) were dissolved in dimethyl sulfoxide (DMSO) and stored at −20 ☌. LLOME (Sigma, L7393) was dissolved in ethanol and stored in aliquots at −20 ☌. All cells were maintained at 37 ☌ with 5% CO 2. Both media were supplemented with 8% fetal bovine serum (FBS) and penicillin/streptomycin. 293T, U2OS, COS7, and BJ cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) PC3 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640.
#Noti enzyme free#
All cell lines used in this study were free from mycoplasma contamination based on PCR detection and were regularly maintained with mycoplasma reagent. Cell lines in this study have different morphologies and growth rates and contamination were constantly monitored. 293T, U2OS, PC3 and BJ cells were authenticated through short tandem repeat profiling and the profiling data are publicly available from ATCC.
If you run out of DNA, do the same procedure all over again, don't think that your BAC sequence will be the same.All cell lines were from ATCC. So, culture a colony, make a mega prep, fingerprint it extensively to make sure that there is no rearrangement and that you are getting all bands right (you may have to do a southern, not just digest), and then use that same DNA for all your expts. This is generally not a big problem, but if your particular BAC has a lot of rearrangment prone sequence, you may get subtle or dramatic changes in restriction pattern. Not from many different colonies, nor from a colony that has been propagated many times over in between DNA preps. Whichever method you use, make sure that in all digest comparsions you are making, you have used a BAC DNA that have all come from a single colony on a plate. I run gel and read nanodrop to identify the BAC quality and quantity then only digest it. I always use the QIAGEN Large Constrcut Kit to extract the BAC DNA.
I hope you are using the same BAC preparation everytime.
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