Biomedical Engineering Reference
In-Depth Information
2.3 Reagents,
Buffers, Compounds,
Dyes, and Antibodies
Methanol, bovine serum albumin (BSA, Sigma-Aldrich, #05470),
and Tween (PBST) were purchased from Sigma-Aldrich (#p1379);
nuclear factor (NF)-
B2 p100/p52 antibody was from Cell
Signaling (4882; Cell Signaling Technology, Danvers, MA); the
secondary antibody ab96921 DyLight 594 was from Abcam,
Cambridge, UK (donkey anti-rabbit IgG (H + L)); and DAPI was
purchased from Sigma-Aldrich (#32670).
Leptomycin B (LC Laboratories, Woburn, MA, USA), fi xa-
tives: Our fi xative of choice is 6 % paraformaldehyde prepared in
PBS at a pH of 7.2-7.4. To prepare 100 mL of paraformaldehyde,
add 4 g of paraformaldehyde powder to 100 mL of PBS and grad-
ually heat to 60 °C under a fume hood. To clear the solution,
slowly add drops of 1 N NaOH while stirring. Do not allow the
temperature of the solution to exceed 65 °C. The fi nal pH can be
adjusted with NaOH or HCl if necessary. Blocking buffer: 1 % BSA
(IgG- and protease-free) in phosphate-buffered saline (PBS) to
block nonspecifi c staining. If there are problems associated with
high background, 5 % normal serum from the host of the second-
ary antibody may be added to the blocking buffer.
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2.4 Cells and Cell
Culture
The human osteosarcoma cell line U2OS was obtained from the
American Type Culture Collection (Manassas, VA). The
U2nesRELOC cells stably express the reporter construct
pRevMAPKKnesGFP, which has been described earlier [
14
]. pRe-
vMAPKKnesGFP carries the NES from MAPK kinase (MAPKK)
cloned between the BamHI and AgeI sites of pRev(1.4)-GFP and
sandwiched between the Rev and the green fl uorescent protein
(GFP) coding sequences.
U2nesRELOC cells are maintained in T-175 cm2 fl asks at
37 °C with 5 % CO
2
in Dulbecco's modifi ed Eagle's medium
(Gibco Life Technologies, #11960-044) and supplemented with
10 % fetal bovine serum (Sigma) and Antibiotics-Antimycotics
(Gibco Life Technologies, #10378-016). Upon reaching about
80 % confl uency (about every 4-5 days), the cells were subject to
passage in the following steps:
1. Wash cells twice with DPBS buffer. This step removes general
debris as well as facilitates cell detachment.
2. Trypsinize the cells using TrypLE™ Express (Gibco Life
Technologies, #12604-021) at room temperature (RT) for
5 min.
3. Gently tap the fl ask and neutralize the TrypLE™ Express with
a cell culture medium.
4. Split a desired volume of cells to a new fl ask or imaging 96-well
plates (Greiner Bio-One #655087).
