Step 1) pilot experiment
N2102Ep confluency and seeding ratio in 96 well plate: seeding :2K, 5K, 10K, 15K, 20K, 25K confluency next morning (visual): 5%, 20%, 30%, 40-50%, 50-60%, 70%
Step 2) diluting drugs with DMSO stockes: 10 mg vials. RPT-000012 +3.413 ml DMSO: 10 mM RPT-000473 +1.855 ml DMSO: 10 mM
Step 3) 1:3 serial dilution
Prepare: 10mM, 3.33mM,1.1 mM, 370uM, 123 uM, 41uM,13 uM, 4uM, 1uM,0
Add 15ul of stock to tube 1 Add 10 ul DMSO to tubes 2-10 Take 5ul of tube 1 and add it to the next tube. Mix (repeat till tube 9).
Step 4) Drug treatment
Note: exclude the corner rows and columns of 96 well plate during seeding to avoid the edge effect while reading the plate.
A- seed the cells (20K cells/well) in 100ul media, let it attach overnight Next day, add DMSO /drug (500X) in 100ul media. Wait for 4 days B- seed the cells (15K cells/well) in 100ul media, let it attach for 6 hours Then, add DMSO /drug (500X) in 100ul media. Wait for 2 days (so they can have enough time to divide). In triplicates, include controls : cells in media only plus wells containing only media (lumin. Background),
Drug dilutions (10 for each drug) in DMSO should be diluted in media 1000X (we can do it stepwise, first dilute it in the media 100X and then 5X, add 100ul to the cells. ( final Dilution 1000X, 0.1% DMSO in media)
Step 5) Cell titer glo
Wells containing 20K were confluent and the wells containing 15K were almost 70-80% confluent.
Thaw cell titre overnight 4 °C overnight, then water bath 22 °C, mix gently Equilibrate plate to RT, 30 min, Aspirate 100ul of media. Add same volume cell titer (100ul for 96 well plate), mix 2 min, orbital shaker (cell lysis) 10 min RT(stabilize signal).
ATP standard ATP Mwt: 605. Prepare 1uM ATP in media Prepare serial 10X dilution > 1uM, 100nM, 10nM, 1nM, 0.1nM Add cell titer (100ul), mix 2 min shaker, record after 10 min
Step 6) Glow max
Turn on>Home> instrument control>PMT activation (5min)>cell tiger glow 2 protocol Plate >select all wells> Door> place microplane Start> Edit(file name)> OK
Calculate: ATP content +/- SD (n=3 replicates)
Graph : fold of growth /control (no treatment)
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