20.109(F13): Mod 2 Day 7 HTS and Analysis

Introduction

Today is the big day!

Note why we use EDTA versus trypsin/EDTA for this protocol.

Part 1: Set-up BRET Assay Plate

Please read all the way through this protocol before starting your work!

1. Two datasets will be obtained today:
   1. A dose-response (DR) curve of growth factor vs. SH2-EGFR interaction -- the growth factor concentration will vary (no drug).
   2. A dose-inhibition (DI) curve of inhibitor vs. SH2-EGFR interaction -- the drug concentration will vary (constant growth factor).
2. Set-up 6 eppendorf tubes for each dataset as follows:
   • Label each tube and add the indicated volume of BRET Buffer (BB) to the tubes labeled DR1-6 only.
   • The final concentration of growth factor and inhibitor will be used later for your calculations.

Tube label	Final GF Conc (μM)	Vol of BB (μL)	Tube label	Final GF Conc (μM)	Final Inhib Conc (μM)	Vol of BB + GF (μL)
DR1	100	450	DI1	100	10	450
DR2	10	405	DI2	100	1	405
DR3	1	405	DI3	100	0.1	405
DR4	0.1	405	DI4	100	0.01	405
DR5	0.01	405	DI5	100	0.001	405
DR6	0	405	DI6	100	0	405

3. Obtain your growth factor and inhibitor of interest from the front bench.
   • All growth factors are at a stock concentration of 30 mM.
   • All inhibitors are resuspended in DMSO at a concentration of 30 mM.
4. Prepare the dose-response tubes by diluting 10x into each tube:
   • Add 4.5 μL of growth factor to tube DR1.
   • Mix by vortexing.
   • Remove 45 μL from DR1 and put into DR2
   • Mix by vortexing.
   • Repeat until you get to DR5.
   • Do not add growth factor to DR6
5. Prepare the growth factor solution for the dose-inhibition dataset:
   • Add 30&mu:L of growth factor to 2.7 mL of BRET Buffer
   • Add the volume of BB + GF to each DI tube as indicated in the above chart
6. Add the inhibitor to the dose-inhibitor tubes by diluting 10x into each tube:
   • Add 4.5 μL of inhibitor to tube DI1.
   • Mix by vortexing.
   • Remove 45 μL from DI1 and put into DI2
   • Mix by vortexing.
   • Repeat until you get to DI5.
   • Do not add inhibitor to DI6
   • Instead of inhibitor, add 4.5 &mu:L of DMSO to DI6 (find DMSO on the front bench)
7. Pick up an opaque 96-well plate from the front bench. This will be your assay plate.
8. Follow the plate map below to add growth factor and inhibitor to the appropriate wells:

	1	2	3	4	5	6	7	8	9	10	11	12
A	DR6 - SH2+EGFR	DR6 - SH2+EGFR	DR5 - SH2+EGFR	DR5 - SH2+EGFR	DR4 - SH2+EGFR	DR4 - SH2+EGFR	DR3 - SH2+EGFR	DR3 - SH2+EGFR	DR2 - SH2+EGFR	DR2 - SH2+EGFR	DR1 - SH2+EGFR	DR1 - SH2+EGFR
B	DR6 - SH2 only	DR6 - SH2 only	DR5 - SH2 only	DR5 - SH2 only	DR4 - SH2 only	DR4 - SH2 only	DR3 - SH2 only	DR3 - SH2 only	DR2 - SH2 only	DR2 - SH2 only	DR1 - SH2 only	DR1 - SH2 only
C	DR6 - RLuc+EGFR	DR6 - RLuc+EGFR	DR5 - RLuc+EGFR	DR5 - RLuc+EGFR	DR4 - RLuc+EGFR	DR4 - RLuc+EGFR	DR3 - RLuc+EGFR	DR3 - RLuc+EGFR	DR2 - RLuc+EGFR	DR2 - RLuc+EGFR	DR1 - RLuc+EGFR	DR1 - RLuc+EGFR
D	DI6 - SH2+EGFR	DI6 - SH2+EGFR	DI5 - SH2+EGFR	DI5 - SH2+EGFR	DI4 - SH2+EGFR	DI4 - SH2+EGFR	DI3 - SH2+EGFR	DI3 - SH2+EGFR	DI2 - SH2+EGFR	DI2 - SH2+EGFR	DI1 - SH2+EGFR	DI1 - SH2+EGFR
E	DI6 - SH2 only	DI6 - SH2 only	DI5 - SH2 only	DI5 - SH2 only	DI4 - SH2 only	DI4 - SH2 only	DI3 - SH2 only	DI3 - SH2 only	DI2 - SH2 only	DI2 - SH2 only	DI1 - SH2 only	DI1 - SH2 only
F	DI6 - RLuc+EGFR	DI6 - RLuc+EGFR	DI5 - RLuc+EGFR	DI5 - RLuc+EGFR	DI4 - RLuc+EGFR	DI4 - RLuc+EGFR	DI3 - RLuc+EGFR	DI3 - RLuc+EGFR	DI2 - RLuc+EGFR	DI2 - RLuc+EGFR	DI1 - RLuc+EGFR	DI1 - RLuc+EGFR

9. Once all wells have been filled with the appropriate contents, cover the plate with parafilm to prevent evaporation.

Part 2: Prep cells for BRET Assay

The cells that you transfected last time have been serum starved overnight to reduce any background SH2-EGFR interactions due to the presence of growth factors in serum. Similar to the cell stimulation done on M2D4 for our Western blot analysis, all steps will be completed in the main lab -- no sterile technique is required today.

1. Grab your 3, 60mm dishes of CHO-K1 cells transfected last time from the TC incubator and bring them to your lab bench.
2. You can find PBS and 0.5 mM EDTA on the front bench.
3. Aspirate the cell culture media from each plate.
4. Rinse your cells carefully with 3 mL of PBS -- remember to aim at the side of the plate and not directly towards the cells!
5. Remove the PBS and add 0.5 mL of 0.5 mM EDTA to each plate.
6. Bring the plates to the 37C incubator at the back of the lab (yes, the one that you used for bacterial culture) and incubate for 5 min.
7. During your 5 min incubation, prepare 3 - 15 mL conical tubes to spin down your cells and 3 - 1.5 mL tubes for cell counting.
8. After the 5 min incubation:
   • Add 3.5mL of BRET buffer to each plate and resuspend the cells by triterating well.
   • Add the total volume of 4mL cell suspension to the pre-labeled 15 mL conical tubes.
   • Save 20 μL of cell suspension -- put into the pre-labeled 1.5 mL tubes.
   • Put your 1.5 mL tubes in the ice bucket at the front of the room.
9. Spin the 15 mL conical tubes in the large centrifuge near the TC room door at 300xg for 5 min to pellet your cells.
10. Carefully remove the supernatant from your cell pellet.
11. Resuspend your cell pellet in 1.25 mL of BRET buffer.
    • Hint: Add 2 x 612.5 μL using your pipetman.
    • Take care not to introduce air bubbles into your cell suspension.

Part 3: BRET Assay

At this point in the day, time becomes important. The peak in EGFR phosphorylation occurs between 5 and 15 min after the addition of growth factor. Therefore, it will be important to add your cells quickly, but with care to not introduce air bubbles. Make sure you've read through the next section before starting!

1. Have your lab timer ready to go and set to zero. Instead of counting down, we will count up to keep track of assay timing.
2. Inform the teaching staff that you are ready to start the assay. This is an important step because the RLuc substrate will be made fresh for each team.
3. Remove the parafilm from the top of your 96-well plate.
4. Add 50 μL of cell suspension to the appropriate wells.
   • You and your partner may want to alternate adding cells to complete this job faster.
   • Make sure to pipette up and down several times before starting to mix the cells. You may want to pipette up and down a few times before each addition.
5. Once all the cells have been added, start your timer.
6. After 5 min, bring your plate to the plate reader room with the teaching staff.
7. After 8 min, add 50 μL of 30 μM choletarzine to each well using the multichannel pipette.
8. After 10 min (2 min post-choletarzine addition) start reading the fluorescence (530 nm) and luminescence (485 nm) from each well.
9. Collect your data and analyze!