Prepare plasmids for the experiment.
Step 2: Cell culture
A) HEK293 cells are cultured in a 10cm dish with DMEM-high glucose medium supplemented with 10% FBS, 2mM Glutamine, 50unit-50µg/ml Penicillin-Streptomycin at 37˚C with 5% CO2.
B) One day before transfection, re-plate the HEK293 cells into 35mm dishes.
1) Prewarm culture medium in a 37˚C water bath. Thaw the 2.5% trypsin stock in room temperature.
2) Aspirate culture medium from the 10cm dish with HEK293 cells. Gently rinse cells with about 2-3ml Versene once. Aspirate the Versene and add 1ml Versene containing final 0.05% trypsin to the cells. Gently rock the plate and let the trypsin solution cover the whole plate. Leave the plate in the cell culture incubator for 5 min.
3) When the cells are fully detached from the dish, add 1ml warm culture medium into the dish. Gently pipette up and down to disperse the big cell clumps until all the cells are dissociated. Transfer the cells to a 50ml conical tube and centrifuge it at 800rpm for 4 min in a swinging-bucket centrifuge.
4) Discard the supernatant and resuspend the cell pellet in fresh culture medium. Gently resuspend the cell pellet to single cell suspension.
5) Count the cells using a Bürker chamber.
6) Plate 400,000 cells, 2ml medium to one 35 mm dish. Based on the number of plasmids needed to be transfected, prepare one more dish for untransfection control.
Step 3: Transfection
The day after plating, transfect the cells using Calcium-phosphate precipitation.
1) Total 2.5µg plasmid DNA is used for one 35mm dish. Total volume of DNA mix is 62.5µl including 6.25 µl 2.5M CaCl2. 2XHebs stock is thawed and 62.5 µl is used for each transfection.
2) Drop the DNA and CaCl2 mix into the 2XHeBs while swirling and wait for 30mins before distributing the complex dropwise evenly over the cultures.
3) Leave the transfected cells in the incubator for 48 hrs. The dish for the untransfected control is not touched but remains in the incubator the same time as the transfected ones.
Step 4: Prepare cell lysate
1) Pre-cool 1x PBS in ice. Thaw the LSB 10x stock and protease inhibitors. Prepare 1xLSB supplemented with 10µg/ml Leupeptin, Aprotinin, Pepstatin A, 100µg/ml PMSF, 1mM DTT, 0.5% IGEPAL® CA-630. Cool down the solution in ice.
2) Quickly aspirate cell culture medium from the 35mm dish and add ice-old PBS towards the inner side of the dish to rinse the cell layer without disturbing it. Leave the dish on ice and gently rock it briefly. Repeat the rinse again. Aspirate the PBS and add 0.5ml supplemented LSB solution. Use a cell scraper to scrape the cells from the dish. Use a 1ml pipette to transfer the lysate into a 1.5ml microfuge tube. Pipette up and down 10 times to further lyse the cells. Use the same procedure for the untransfected dish.
3) Pre-cool the benchtop centrifuge to 4 ˚C. Pre-clear all cell lysates at 4 ˚C, by centrifugation at 20,000g for 10 min. After centrifugation, transfer the supernatant to a new microfuge tube, aliquot the supernatant to smaller volume and snap freeze all the tubes in liquid nitrogen.
Step 5: measurement of relaxation time constant of the cell lysate
1) On the day of measurement, thaw the cell lysate on ice and cover the tubes with foil. Re-centrifuge the cell lysate at 4 ˚C, 20,000g for 10min after thawing.
2) Turn on the plate reader to ready it for measurement.
3) Transfer 20 μl of cell lysate to a half-area 96 well plate. Add 5 μl mineral oil on top to prevent evaporation. Only prepare the samples which are used for current measurement. Leave other samples on ice in darkness.
4) Before each measurement, the plate is equilibrated to either 25°C or 37°C in the plate reader for 5-10min. The stability of the dark state is determined by measurement after temperature equilibration. The adduct state is generated by 30 s continuous exposure to blue LEDs (peak ~465nm) at 2 mW.cm-2 (previously measured at 100% duty cycle with an optical power meter such as x-cite XR2100 and sensor XP750 (Lumen Dynamics) or alternative). The recovery to dark state is quantified as a recovery from dequench by rapidly transferring the plate to the reader and starting the measurement protocol immediately. This protocol consists of measurement cycles set to flying mode (3 xenon flashes per well). The excitation filter (430/10nm) and the emission filter (475/10nm) are used. Only 8 wells are used per measurement to minimize the cycle time (3 s per cycle). A total 20 cycles are measured, corresponding to 20 flashes over 1 minute in each of 3 positions per well. Background signal from 20 μl untransfected cell lysate is subtracted from the raw signal of each plasmid. All cycles are normalized to the first measurement of individual plasmid, and the data are fitted to an exponential decay with Graphpad Prism5.