IceMODULE 3 – Day 2Harvest HeLa cells previously transfected with EGFP and p53 siRNAs; analyze EGFP expression by microscopy and flow cytometry These instructions should look familiar! Note that HeLa cells may be harder to trypsinizeand harvest than the embryonic stem cells you worked with in Module 2.Required Materials:1 six well plate of HeLa cells from Day 1.4 ml Trypsin15 ml serum containing RPMI media10 ml PBS5 ml PBS on ice6 15 ml conical tubes6 12x75 mm polystyrene flow cytometry tubes P1000 w/tipsIceProcedure in lab (read each task entirely before starting): Visually inspect each of the six well plates under the fluorescent microscope to determine whether the expression of EGFP is altered in any of the wells. Take digital pictures of a typical field of cells for each of the six wells. Label six 15 ml conical tubes and six flow cytometry tubes, one of each kind of tube per well. An example of how these could be labeled is as follows: EGFP(1), EGFP (2), p53(1), p53(2), Ctrl (1), Ctrl(2). Pipette off all media from each well into waste (~1 ml per well). Rinse cells by doingthe following: Add 1 ml of PBS to each well (pipette slowly into wells, do NOT pipette forcefully), gently rock plate to rinse wells, and then pipette off PBS into waste.  Detach cells by doing the following: Add 0.5 ml of trypsin per well. Put plate in 37C incubator for 5 min. After 5 min incubation time, look under microscope to see if cells have detached. If they have not detached, gently knock plates on sides and put back in incubator for 1-2minutes. Add 2 ml of media to one well at a time to deactivate the trypsin; add the medium vigorously so that cells become detached. Do not let cells sit in trypsin for longer than necessary. For one well at a time, pipette the cells up and down 5-10 times in order to wash cells off the dish and to break cell-cell contacts and mix cells well. ***Use a P1000 pipettor and make sure to pipette up and down vigorously in the edges of the well.*** Transfer the media plus cells (total volume) to the appropriately labeled 15 ml conical tube.  Pellet the 2.5 ml cell suspension by spinning for 5 min @ 1500 rpm in the 15 ml conical tubes. Make sure lids are seated properly and shut tightly before starting the centrifuge. Since we only have one centrifuge, please make sure that at least two groups are pelleting at the same time. After spinning down cells, pipette off the media, being careful not to dislodge the cell pellet.  Disperse the cell pellet by flicking the tube. Resuspend the pellet in 300ul ice cold PBS. Pipette sample up and down to resuspend cells, and then transfer immediately to polystyrene flow cytometry tubes and put cells on ice. You are ready to go to the FACS center.Flow Cytometry Analysis (for more info: http://web.mit.edu/flowcytometry/www/):1. Turn the Flow Cytometer on. Turn the computer on.2. Get user name and password from TA.3. Get an acquisition file from TA.4. Follow instructions on FACS machine.5. Points to remember for FACS:a. During the setup:1. Make sure to press the “Set” button after selecting the data file in the instrument settings menu.2. Set the analyzer to count 20,000 cells for each sample.b. Run the Negative Control samples first.c. Be sure to flush with water by fitting the water tube over sipper after every sample.d. Print a copy of each sample run.6. Shut down according to the notes on the FACS