50 mL Falcon using sterile serological pipette. Add 5 mL of
heat inactivated FBS using sterile serological pipette and gently
mix the media (seeNote 2). Pipette out 500μL of Pen-Strep
Stock (100, 10,000 U/mL, penicillin and streptomycin) and
add to the above solution. Gently mix this cell culture media
and store at 4C(seeNote 1).- 1μg/μL DOTAP Transfection reagent (Roche,seeNote 3).
- 1Opti-MEM (Reduced Serum Minimum Essential Media):
Opti-MEM is a modification of Eagle’s Minimal Essential
Medium, buffered with HEPES. Aliquot 40 mL of Opti-
MEM in sterile Falcons and store at 4C in the dark for
optimum performance. - AMUTP (60 mM): Prepare 60 mM AMUTP stock solution in
autoclaved water and store at 40 C. Thaw it on an ice bath
before use. - Transfection mix: In a plastic vial, prepare 50μLof12mM
AMUTP in HBS buffer by mixing 10μL of 60 mM AMUTP
and 40μL of HBS buffer. In another vial, prepare a 100μL
DOTAP solution containing 30μLof1μg/μL DOTAP and
70 μL of HBS. Transfer the 12 mM AMUTP into the above
DOTAP solution and mix well. Incubate this mix at RT for
15 min. After 15 min add 450μL of reduced serum media
(Opti-MEM) and mix gently to generate the transfection mix
(final volume 600μL and 1 mM AMUTP). Prepare this mix in
biosafety cabinet and just before transfection experiments. - 3.7% formaldehyde: Prepare working solution of 3.7 v/v %
formaldehyde solution by diluting 5 mL of 37% formaldehyde
in 45 mL of 1PBS in 50 mL sterile Falcon. Store 3.7%
formaldehyde solution at room temperature. - Triton X 0.5% solution: Prepare a primary stock of 10 w/v %
Triton X-100 solution by dissolving 1 g of Triton X-100 in
8mLof1PBS in 15 mL sterile Falcon. Mix stock solution
carefully by stirring the Falcon on a rotor to avoid frothing (see
Note 4). Add 1PBS to make 10 mL stock solution of 10%
Triton X-100 and mix well. Prepare 50 mL working solution of
0.5% Triton X-100 by diluting 2.5 mL of 10% Triton X-100
using 45 mL 1PBS. Store 0.5% Triton X-100 solution at
room temperature. - Live cell imaging media: Prepare live cell imaging medium
using DMEM (Life Technologies, Catalog No. 31053028) in
10% FBS. Take 45 mL of plain DMEM in a sterile 50 mL
Falcon and add 5 mL of heat inactivated FBS using sterile
serological pipette and gently mix the media. Store this live
cell imaging medium at 4C(seeNote 1). - (Optional) 2μM actinomycin D in Opti-MEM (seeNote 5).
362 Anupam A. Sawant et al.