MODULE 3 – Day 2
Harvest 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 trypsinize
and 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 Trypsin
15 ml serum containing RPMI media
10 ml PBS
5 ml PBS on ice
6 15 ml conical tubes
6 12x75 mm polystyrene flow cytometry tubes
P1000 w/tips
Ice
Procedure 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 doing
the 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 1-2
minutes.
 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.
2.
3.
4.
5.
Turn the Flow Cytometer on. Turn the computer on.
Get user name and password from TA.
Get an acquisition file from TA.
Follow instructions on FACS machine.
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 machine.