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

Introduction

Today is the big day! Today we are incredibly lucky to be able to complete our module using the High Throughput Screening facility in the Koch Institute. Unfortunately, our screen doesn't require the robot -- but we will use a very advanced plate reader with the help of a former 20.109 TA, Ian Tay.

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

Part 1: Prep assay plate for BRET assay

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

Two datasets will be obtained today:

• A dose-response (DR) curve of growth factor vs. SH2-EGFR interaction -- the growth factor concentration will vary (no drug).
• A dose-inhibition (DI) curve of inhibitor vs. SH2-EGFR interaction -- the drug concentration will vary (constant growth factor).

We will use DR and DI shorthand a lot today, so remember what they mean!

Plate set-up:

1. Grab 5, 15 mL conical tubes from the front bench.
2. Prepare 6 eppendorf tubes for the dose-response dataset as follows:
   • Label each tube and add the indicated volume of BRET Buffer (BB) to the tubes labeled DR1-8.
   • 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)
DR1	100	980
DR2	20	800
DR3	4	800
DR4	0.8	800
DR5	0.16	800
DR6	0.03	800
DR7	0.006	800
DR8	0	800

3. Obtain your growth factor and inhibitor of interest from the front bench.

• All growth factors are at a stock concentration of 10 mM.
• All inhibitors are resuspended in BRET Buffer at a concentration of 1 mM.

4. Prepare the dose-response tubes by diluting 5x into each tube:

• Add 20 μL of growth factor to tube DR1.
• Mix by vortexing.
• Remove 200 μL from DR1 and put into DR2
• Mix by vortexing.
• Repeat until you get to DR7.
• Do not add growth factor to DR8

5. Prepare the growth factor solution for the dose-inhibition dataset.

1. • Label six eppendorf tubes DI1-8.
   • Add 30 μL of growth factor to 2.97 mL of BRET Buffer
   • Add the volume of BB + GF to each DI tube as indicated in the chart below:

Tube label	Final GF Conc (μM)	Final Inhibitor Conc (nM)	Vol of BB + GF (μL)
DI1	100	10,000	294
DI2	100	2000	240
DI3	100	400	240
DI4	100	80	240
DI5	100	16	240
DI6	100	3.2	240
DI7	100	0.64	240
DI8	100	0	240

6. Add the inhibitor to the dose-inhibitor tubes by diluting 5x into each tube:

• Add 6 μL of inhibitor to tube DI1.
• Mix by vortexing.
• Remove 60 μL from DI1 and put into DI2
• Mix by vortexing.
• Repeat until you get to DI7.
• Do not add inhibitor to DI8

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 50 μL growth factor or inhibitor to the appropriate wells:

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 4, 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 4 - 15 mL conical tubes to spin down your cells.
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.
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 1.8 mL of BRET buffer.
    • Hint: Add 2 x 900 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. First, we will take our cells and other supplies across the courtyard to the High Throughput Screening facility at the Koch Institute. Once there we will finish our assay.

Note: 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!

Once we get over to the Koch:

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 Viviren 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. Put the remaining cells in the ice bucket.
7. After 10 min, inform Ian and Kim that you are ready to scan.
8. After 12 min, add 50 μL of 30 μM Viviren substrate to each well using the multichannel pipette.
9. After 15 min (3 min post-Viviren addition) start reading the fluorescence (530 nm) and luminescence (485 nm) from each well using the Tecan plate reader.
10. Collect your data and analyze!