Analysis of Receptor–Receptor Interaction by Combined Application of FRET and Microscopy - Methods in Cell Biology - page 247

14.2.3.4 Protease inhibitors and phosphate-buffered saline stock
solutions
PMSF inhibitor : Prepare 50 ml of 100 mM stock solution in 70% ethanol and store
them in 500 20 C. m l aliquots at

CLAP100 inhibitor : Prepare separate stock solutions of each organic

compound and store them at 20 C: leupeptin (10 mg/ml in distilled water),

chymostatin (10 mg/ml in DMSO), antipain (10 mg/ml in 70% ethanol), and
pepstatin (1 mg/ml in methanol). To prepare 50 ml of solution, mix all the
compounds in 50 ml of 70% ethanol and prepare 500 m l of aliquots and sterile

20 C. Thaw before use.
Amino acids : Prepare 200 ml stock solution of each amino acid (1.050 g of
L -cystine, 0.712 g of L -alanine, 1.060 g of L -aspartic acid, 0.920 g of L -proline,
1.350 g of L -glutamic acid, and 1.351 g of L -asparagine) and store as 10 ml
aliquots at

filter it before storing at

20 C.

10 phosphate-buffered saline (PBS) : Combine 80 g of NaCl, 14.195 g of

NaHPO 4 , 2.0 g of KCl, and 2.4 g of KH 2 PO 4 and dissolve them in 1 l of distilled
water. Autoclave the solution and later adjust the pH to 7.4 with 1 MNaOH. Store
the stock solution at room temperature for 2 months. Dilute 10 times before using
for experiments.

14.3 METHODS (PROTOCOLS AND PROCEDURE)
14.3.1 Cell preparation and transfection timing
14.3.1.1 Culture of cells
Start new culture: Resuspend the frozen cells in 1 ml of DMEM media and add the
suspension into a flask containing 19 ml of DMEM media.

14.3.1.2 Reculture and seeding cells
Neuroblastoma cells (N1E-115 cell lines)

1. Put a vial of fresh media DMEM (FCS/PS) into the water bath at 37 C for
15 min.
2. Pour 6 ml of media per petri plates.
3. Pour the old media out of the plate.
4. Add 5 ml of fresh media into the plate and detach the cells and separate the cells
by gently sucking in and out 3-4 times. Avoid forming bubbles.
5. Pour 1.5 ml of the cells into the new petri plates and 500

m l for 6-well and 100 m l

for 12-well (including glass coverslips on the bottom for microscopes) plates.
6. Let the cells grow normally for 2 days or one for transfection at 37 C/5% CO 2 .

14.3.1.3 Transfection
1. Calculate the volume of Opti-MEM, Lipofectamine, and vectors (depending on
concentration) to be used according to manufacturer's instruction.