Analysis of Receptor–Receptor Interaction by Combined Application of FRET and Microscopy - Methods in Cell Biology - page 246

Application of FRET and Microscopy

￿ TIRF microscope (TILL Photonics):
￿ iMIC platform with extensions for TIRF and FRET measurements (TILL
Photonics): iMIC microscope with a polytrope and dichrotome dual emission
extension.
￿ Laser lines: 440 (50 mW), 488 (50 mW), 515 (40 mW), and 640 (50 mW) from
TOPTICA Photonics for CFP-YFP and GFP-mCherry derivatives FRET pairs.

￿ /1.46 Oil TIRF (Zeiss).

￿ Two MBSs MBS405/488/561 and MBS400/514/561/640 (AHF
Analysentechnik).
￿ Filterset I (dichroic 515dcxr, CFP emitter 482/18 nm, and YFP emitter514/
3 nm) and filterset II (560dcxr, GFP emitter 561/14 nm, and mCherry emitter
655/40 nm).
￿ CCD camera for recording simultaneous two emission channels.
￿ Oligochrome rapid filter switch device with xenon (150 W).
￿ Multichannel mode of the live acquisition imaging software.
￿ Custom-built chambers.

a -Plan-Apochromat 63

14.2.3 Reagent setup
14.2.3.1 Preparation of cell culture media
1. To prepare 1 L of DMEM: First dissolve 13.4 g of powdered DMEM in 100 ml of
distilled water. Then, add 1.81 g of HEPES, 2.53 g of NaHCO 3 and bring the
volume up to 800 ml by adding water. Then, add 10 ml of each amino acid and
adjust the pH to 7.4 with NaOH and bring the final volume to 1 l. Sterile filter the
medium and store at 4 C up to 1 month.
2. Take bottle of the DMEMmedium (in cooling room) and put in water bath (37 C).
3. Take one flask of frozen FCS (fetal bovine serum) and one flask of frozen S/P
(penicillin-streptomycin) and put as well in water bath.
4. Add thawed 10% FBS and 1% S/P (5 ml) to a 500 ml DMEMmedium and mix it.
5. Sterile filter the media and store in a refrigerator (4 C).

14.2.3.2 Buffer A for cell lysis
Combine 10 mM HEPES, 150 mM NaCl, and 1% Triton. We usually prepare 1 M of
HEPES and NaCl stock solution and keep them for 2 months. To prepare 500 ml of
buffer A, weigh HEPES and NaCl according to their molecular weight and add
500 ml of double-distilled water. Adjust the pH to 7.4 with 1 M NaOH. Filter the
buffer before use. The buffer can be stored at 4 C and is stable for 2 months.

14.2.3.3 Buffer B for FRET imaging and Fluorolog experiments
Combine 150 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 2 mM CaCl 2 , and 10 mM
HEPES. Adjust the pH to 7.4 with 1 M NaOH. Check the osmolality of the DMEM
medium and the buffer B. Bring the osmolality of the buffer same as the medium by
adding D þ glucose, which is generally around 342 mOsmol before starting of FRET
measurements.