Christina Mendoza’s Summer Science
Research Journal 2013
Locasale Lab, Cornell University
134 Kinzelberg Hall
Day 1 with Sam-Monday
1) Looked at breast tissue cells under a microscope.
2) Practiced micro pipetting
3) Added lysis in solution to prevent protein damage,
cutting up, etc. in order to collect protein lysates for
Westerns.
4) Used a cell lifter to scrape
cells of plate and into the lysis
buffer. Then the cells and
buffer were transferred to an
eppendorf tube and incubated
on ice.
5) Put cells in eppendorf
tube centrifuge for 30
minutes at max speed
6) Performed a BioRad Bradford Assay using the
SpectraMax M3 to determine the protein
concentration in each sample lysate for even
loading during westerns.
7) Western Blot to determine the global
methylation/acetylation changes under different
metabolic conditions. Cells were grown in varying
concentrations of glucose.
6/24/13
L
MM-
MM+
5+
Figure 1 from Sam Berkheimer.
Anti-PHGDH raw image. At this point
without a loading control, it looks like the
expression system may be working when
Mcf10a cells are grown in 5mM glucose
supplemented DMEM, -Glutamine, +NEAA.
However, there seems to be something
funny comparing the Mcf10a Media -/+
DOX samples. MM+ should show over
expression of PHGDH, however it looks as
if MM- has more PHGDH. Will need to do
Actin loading control.
Day 2 with Sam-Tuesday
6/25/13
1) Running out of TBST, so we diluted a 10x solution of TBST by adding 900
mg of di H20 to 100 mg of the TBST
2) Washed the product of the gel electrophoresis in TBST to cleanse it of the
dry milk solution for about 10 minutes
3) Note: The primary antibody was added last night by Sam
4) The gel is being mixed with TBST on the rocking platform
5) Added the secondary antibody
6) Added stripping buffer to the membrane and
put it in the incubator (down the hall) for 30
minutes at 60°C
Does it always rain this
much in Ithaca?
Reading an article published by Professor Locasale
in my free time
Figure 1 from Sam Berkheimer. Anti-Beta Actin (Mouse). The signals are from a previous
blot using Mouse Anti-PHGDH. Although I stripped the signal still remained. B Actin (42kD)
is observed around 40-43kD based on ImageLab analysis and is indicated by the arrow.
NORMALIZATION
MM-
MM+
5+
Actin
5920928
5195564
4847180
Intensity
Intensity Norm
PHGDH
PHGDH
Actin Norm
PHGDH Norm
Fold Change
43262336
1.00
43262336
1.48
50925792
27343845
1.22
1.07
62206880.66
29309144.02
2.12
1.00
FIGURE 2 from Sam
Berkheimer. Normalized
PHGDH Fold Change under
doxycycline induction in
Mcf10a cells. Protein levels
were normalized to beta actin.
The results were not expected,
they suggest that addition of
dox actually decreases protein
expression. Normal conditions
(Mcf10a media without DOX)
had 2-fold higher expression of
the PHGDH protein...?
Day 3 with Sam-Wednesday
6/26/13
1) Went to a Bio Laboratory Products Sale with Sam and
Carlie at 10 AM.
2) Looked at cells under a microscope
3) Harvested actin to save for future use (good for up to
two weeks)
4) Rinsed proteins in TBST twice, changed with fresh
TBST every 15 minutes
5) Added NaN3 to save the actin (NaN3 is a
preservative)
6) Took pictures of the protein, focusing on the actin
7) Going to split cells later because they need more
room to grow
8) Practiced some basic skills under the Laminar Flow Hood such as
pipetting up and down, removing old cell media with a suction, always
keeping foot on the pedal before getting tips with suction, and cleaning the
hood well with 70% alcohol
9) Add trypsin to prevent breast tissue cells from building matrixes to the
plate and to each other. This makes it easier to harvest and collect
10) Generally add 9 mL of cell media to petri dishes
11) Basic steps to split cells under the Laminar Flow Hood:
1. Heat up cell media, re-suspension media and trypsin in
the water bath (37°C)
2. Under the hood, clean up using 70%
alcohol spray.
3. Remove old media from plates with a
tip and suction
4. Add 5 mL of sterile PBS to wash out
any left-over media
5. Remove PBS using a tip and suction
6. To trypsinize the cells, add 1 mL of
trypsin, swish around the liquid as
pictured in the
diagram below.
7. Remove most of trypsin but leave a little bit.
Then return cells to the incubator. (5-10
minutes) You will know when the time is right
to continue because the cells will appear to
smear off the plate.
8. Add 1 mL of media to scrape and collect cells
from plate with a micropipette. Transfer liquid
from plate to a 15 mL tube.
9. Add another mL of media to make sure that
you get most of the cells off the plate. Collect
liquid and transfer to the 15 mL tube.
10. Put the tube in a centrifuge for 5 minutes at
1000 PCF. Make sure that there is a good pellet.
11. Remove most of the media from the 15 mL tube
using a tip and suction, and make sure you don’t
suck up the pellet of cells on the bottom.
12. Add 1 mL of media to re-suspend cells, and then
pipet up and down to break the pellet.
13. Add 7 mL of media to the 15 mL tube, and
pipet up and down to evenly distribute the cells in
the liquid.
14. Prepare your new sterile plates- Make sure
they are labeled with the type of cells, cell
media, date, initials, and what passage they are
in (how many times the cells have been split).
15. Add 9 mL of media to each plate.
16. Using a micropipette, add 1 mL of cells from
the 15 mL tube to each plate.
17. Gently swish around cells and then put the
plates in the incubator.
18. Clean up using 70% alcohol spray.
Day 4 with Sam-Friday
6/27/13
1) Looked at split cells from yesterday. They are growing fine, covering
about 30% of the plate.
2) We have to wait until they cover more of the petri plate before we can
split them again.
Day 5 with Sam-Friday
6/28/13
10:19- Looked at 6 cells that were plated on 6/26. 5 are growing fine but
one plate is barely growing, in comparison to the others.
11:35- Heated up media in the water bath to 37°C (body temperature) for
30 minutes
12:00- Made new Mcf10a media by adding Insulin, DMEM, HS, EQF,
Hydrocord, and Cholera. These substances are necessary to add because
these specific breast tissue cells grow best under these conditions.
12:30-Removed old media out of petri dishes,
added 5 mL of PBS to wash out old media
(because there’s a chemical already in the media
that inactivates trypsin), removed PBS, added 1
mL of trypsin and swished it around, then
removed out most of the trypsin but
left a little. Wait about 20 minutes.
1:00- Added media to scrape off the
plates then put it in a 15 mL tube to
be sent to the centrifuge. After 5
minutes, removed the old media so
that just the pellet is left, then added
1 mL of media to re-suspend the
pellet. Then added 7 mL of media and
evenly distributed 1 mL of cells to
each of the petri dishes.
Day 6 with Sam-Monday
7/1/13
10:30- Looked at cells, which are at Passage 3 (split on 6/28/13). The
dishes look crowded so we are going to split them again around 2:00
3:50-6:30- Under the hood:
• Split cells for stock (saved the leftovers with a preservative and put
them in the freezer). The cells that I split are labeled “Mcf10a P4
CM 7/1/13)
• Split cells (from Curry) to be used as a control for the experiment.
These normal muscle cells should die when antibiotics are added
because they don’t express PHGDH.
• Preparing set-up to re-do the experiment with actin, because the
results did not make sense. In the previous experiment, it is highly
likely that a human error occurred. First culture cells in normal
Mcf10a media on 10 mm petri plates to ensure that cells will grow
on the plate. Later the cells will be put into the different media with
varying amounts of glucose.
Day 7 with Sam-Tuesday
7/2/13
1:30- Looked at cells under the microscope, all are growing well around 30%
covering the plate. Planning on changing the media to the varying glucose
amounts for the petri dishes with 6 wells at 2:00. Going to harvest the proteins
at 4, because that would mean that they have been in culture for 24 hours.
2:00- Switched media with the new varying amounts of glucose for the cells
growing in the petri dish with 6 wells.
4:00- Put PBS in 3 cell plates, then removed it. Added 80% Methanol to
each petri dish. Individually put petri dishes in a rubber container filled with
liquid nitrogen. After that, dishes were put into a bucket of dry ice, and then
brought downstairs into a freezer that is -80°C. We want everything to be as
cold as possible because even though the cells are dead, we don’t want the
proteins to denature and fall apart.
4:30- Removed media out of plates with 6 wells, then put them in dry ice.
Afterwards, we put them into the freezer at -80°C.
5:00- Used cell lifters to scrape off cells from each well, and put them into
separate eppendorf tubes. Afterwards, put all eppendorf tubes into the
centrifuge.
5:25-5:37- Micropipetted liquid (metabolites) and not the pellet and
transferred the metabolites to new labeled eppendorf tubes and put them in the
-80°C freezer overnight.
Day 8 with Sam-Wednesday
7/3/13
1:15- Imaging cells at next-door lab and kept others in the incubator.
Heated up media in the water bath (37°C) and going to split cells again
around 2:00.
2:00-2:30Removed media from cells, add PBS to cleanse, remove PBS,
trypsinize cells, then remove most of trypsin. Returned cells
to incubator.
• Removed media from cells, add cold sterile PBS, remove
PBS, put plates in liquid nitrogen, then transferred to a
box filled with ice, then transferred to the -80°C freezer
• For fun, froze a flower in excess liquid nitrogen and
threw it at the ground with Sam and Zheng and
watched it shatter.
2:35- Added 1 mL of media to collect cells from plate, and
another 1 mL to get all cells. Put tube into the centrifuge for 5
minutes at 1000. Removed old media with suction, and then added 1 mL of
media to re-suspend the cells, and another 7 ML of media. Added 9 mL of
media to the petri plates then added 1 mL of media to each plate after
mixing the cell solution (pipetting up and down)
•
**Note to self: Make sure you check cells tomorrow morning because they
are most likely contaminated. If so, tell Ahmad and he will take care of it.
236 Kinzelberg Shared Tissue Culture Facility
Day 9 with Ahmad
7/4/13
10:00-10:10- Looked at cells under microscope (light is usually green but
today it was white). The petri dishes are 30% covered and look okay and not
cloudy, and confirmed by Ahmad.
10:30- Heat up media in water bath for Ahmad’s cells at 37°C
11:00- Watched Ahmad split his liver cancer cells under the hood
Day 10 with Ahmad
7/5/13
10:15- Looked at cells I have been growing under the microscope and they
are covering about 60% of the plate. We might have to split them later.
2:00-4:00- First Lab meeting today in Savage Room 403!
White Board Paint!
Day 11 with Ahmad-Monday
7/8/13
10:15- Looked at my cells that Ahmad split from yesterday 7/7/13. They
are growing and covering less than 10% of the plate. In the incubator, they
are labeled as “P6 CH Mcf10a 7/7/13”
11:00-New graduate student Neel came to the Locasale Lab for his first day
11:36- Observed Ahmad performing a Western for his proteins, and
transferring them.
3:00- I helped add 1 gram of BAS to 1 gram of dry milk under the hood to
two separate
15 mL tubes. We will image them tomorrow.
5:00- Added primary antibody to the dry milk and BSA solution, then put it
in the cold room overnight (walk in fridge).
Day 12-Tuesday
7/9/13
10:10- Looked at my cells that Ahmad split on 7/7/13. They are still
growing and covering less than 10% of the plate.
11:40- Washing product of gel electrophoresis in TBST every 5 minutes on
the rocking platform
2:30- Imaging protein using Bio Rad machine at next door lab
Day 13-Wednesday
7/10/13
10:11- Looked at my cells that were split
7/7/13, under the microscope. They are
now covering about 25% of the plate.
Day 14 with Zheng-Thursday
7/11/13
10:50-Looked at cells under the microscope that were split 7/7/13. They are
now covering about 50% of the plate. I plan on splitting them either
tomorrow or on Monday.
At 1:00, I am going to
watch Zheng administer
drugs to his cells. On 8
different cell lines, he is
going to add these drugs in
different concentrations:
• IA- iodoacetic acid
• DCA- dichloroacetic
acid
Concentrations: 0, .1, 1, 5,
10, 50, 100 micromolars (µm)
The cells are growing in 96 well plates in normal
cell media and then we are going to change the
media and add drug concentrations and grow
the cells for 48 hours. He is going to calculate
IC50 (toxicity of drug to cells). Zheng chose
these drugs because if they act together, there
might be compounded effects because these
drugs both block similar pathways in glycolysis,
but at different points.
1:20-Watched Zheng under the hood, remove
old media from 96 well plates and add the different diluted concentrations of
the drug to each well. I also looked at some of the 8 different cell lines under
the microscope, one of which is colon cancer cells.
Day 15 with Ahmad
7/12/13
10:45-Looked at cells under the microscope that were split on 7/7/13. They
are now covering about 85% of the plate and should be split on Monday.
2:00-3:30-Second Lab Meeting Today-Professor Locasale gave a new talk
concerning glucose modeling/Warburg Effect, entitled “Rewiring of Glucose
Metabolism in Cancer.” This talk is going to mimic the talk he is going give
in Colorado the next week.
Day 16 with Ahmad-Monday
7/15/13
10:30-Looked at my cells under the
microscope split 7/7/13. They are overcrowded and must be split today. Also
the media is starting to turn a tint of
yellow. Change in color is bad and
signals that the cells are using up all the
nutrients and the PH of the solution is
changing, and these conditions can be
harmful to growing cells.
2:30-3:30-Split my Mcf10a cells under
the Laminar flow hood while wearing a
lab coat, with the supervision of
Ahmad. I followed the same procedure
as written out in Day 3.
Day 17-Tuesday
7/16/13
10:45-Looked at my cells under the microscope that were split on 7/15/13.
They are now “Mcf10a P7 7/15/13 CM” and are covering about 5% of the
plate.
11:30-12:30-Working on “Laboratory Safety 2009” Training at Mann
Library
Day 18-Wednesday
9:00-10:30-Attended “Bloodborne Pathogen Lab Training” in
the Vet Education Center with my sister Carlie and Zheng,
which was taught by Instructor Frank A. Cantone.
7/17/13
10:45- Looked at my cells under a microscope that were split on 7/15/13
P7. They are growing and covering about 10% of the plate. Also took a look
at Sam’s cells from “Curry split 7/14/13” and they are growing about 60%
Day 19-Thursday
7/18/13
11:00-Looked at my cells under a microscope that were split on 7/15/13 P7.
They are growing and covering about 20% of the plate.
Day 20-Friday
7/19/13
10:30-Looked at my cells under the microscope that were split 7/15/13 P7.
They are now covering about 20% of the plate. Also, Sam’s Curry cells P7
need to be split because they are overcrowded and the media turning
yellow, this color change indicates a depletion of nutrients for the cells.
Day 21-Monday
7/22/13
10:45-Looked at my cells under the microscope that were split 7/15/13 P7.
They are now covering about 25% of the plate.
Day 22-Tuesday
7/23/13
10:45-My cells as well as Sam’s cells are not in the Locasale incubator.
Going to ask around to find out what happened to them.
2:00-Found out that Ahmad cleaned the
incubator.
Day 23-Wednesday
7/24/13
10:45-Looked at my cells under the microscope that were split 7/15/13 P7.
The cells aren’t looking too good- it’s been over a week since they were last
split and there is barely any growth and there are a lot of floating dead cells
in the media. It is possible that this is due to the fact that I waited too long
to split the cells from P6 to P7.
Day 24 with Sam-Thursday
10:00-11:30, 12:00-4:00-Working on the Pipetting Lab
Lab Protocol: Pipetting
• With H20- 5 µl increments
0  1000 µl
7/25/13
1.
2.
3.
4.
Pipette 5 µl H20, repeat
Weigh tube every 10 times
Record weight
Repeat until you reach 1000 µl (equal to 1mL)
1. Plot weight vs. volume
2. Compute linear regression coefficient
Repeat 3 times with H20 and compute the standard deviation for n=3
measurements.
• Repeat procedure with a more viscous fluid, such as 10% glycerol.
Day 25 with Sam-Friday
7/26/13
10:00-Finished graphs and stat calculations
2:00-Lab Meeting today-Ahmad is presenting
“Metabolic Regulation of Histone Acetylation in Cancer”
2DG Hexokinase (harsh drug, too toxic for cancer
therapy, blocks glycolysis)
Day 26 with Sam-Monday
7/29/13
GROWTH OF CELLS IN SOFT AGAR
Materials
•
•
•
•
6% Agar in Sterile H2O Stock
Cell Culture Media (Warmed to 37C)
6 Well Culture Plates
3% SeaPlaque Agar StockLow gelling temp (26-30C @1.5%)
Methods
Day Before Plating
1. Prepare ~200ml 6% Agar using regular agarose powder and sterile water in a
sterile glass bottle. (Miliq is fine since you are going to boil the mixture anyway
and it will be sterile)
2. Melt agarose mixture by boiling 2x for 30sec each in microwave
3. Under sterile hood dilute the 6% agar mixture 1:10 in order to obtain a final
concentration of 0.6% with cell culture media of choice (+antibiotics) **Note: it
is important that the media is warm otherwise the agarose with solidify before
you can plate it**
4. Add agarose + media mixture (3ml) to each well of a 6 well plate
5. Wrap the plates in plastic wrap and place in 4C for 1 hour (If not using the next
day, plates will keep at 4C for 1 week)
6. Take the plates and place them in the 37C incubator o/n
Day of Plating
7. Prepare at least 200ml of 3% agar solution using Sea Plaque Agarose in sterile
water in sterile bottle (Jason’s from 1/20/2010)
8. Microwave mixture until boiling 2x for 30sec or until perfectly melted
9. For a triplicate experiment, count the cells and prepare a mixture for 4 wells in
7.2ml media(so there is excess)
For example: to plate 100000 cells/well, prepare a mix of a total of
400000 cells in 7.2ml of complete media (warmed to 37C)
10. At the very last moment, add 3% agarose (800ul) using a 5ml pipette for a final
concentration of 0.3% agarose
11. Mix well and plate 2ml in each well trying to be delicate by pipetting in the
center of the well to avoid the bottom from becoming detached from the sides
12. Put the plates at 4C for 1 hour and then place back in the incubator
13. Count colonies after 4 weeks or depending by cell type
Day 27 with Sam-Tuesday
7/30/13
1. Mcf10a (epithelial) cells need contact with something to grow vs. Cancer
cells from Zheng (harsh and fast growing) don’t need contact to grow and
proliferate
*Soft Agar Assay
2. Time microwave vs. solidification and graph temp vs. time to optimize
agar
3. Microwave  37°C (body temp)  add cells
It can’t be too hot or else the cells will die, but you can’t wait too long or
else the gel will solidify before you can add the cells
=test viability/growth, test for anchorage (independent growth) aka can
determine how cancerous a cell is
Then add drugs to cells and run assay again. In theory they should not be
able to grow in the gel for the 2nd assay if the drug is successful and
effective.
3:00-4:00-Watched Sam transfer cholera toxin to eppendorf tubes after
injecting sterile water with a needle, under the hood
Autoclaved bottles with blue cap that we purchased from the stock room
Made 6% solution of agarose- 12 mL agarose powder + sterile water
Microwave 3 mins +30 sec +30 sec to Boil
Solidify overnight
Day 28 with Sam-Thursday
7/31/13
11-12:45-Soft Agar Optimization with the SeaPlaque
2:45-3:30-Soft Agar Optimization with the Lab Agar
Day 29 with Sam-Thursday
8/1/13
(10-3 is the Chromatographic and Mass Spectrometric Training at the
Biotech Building)
1:45-3:15-Attended the afternoon Mass Spec session
Day 30 with Sam-Friday
8/2/13
2:00-Lab Meeting today-Zheng is presenting
1) C13 Isotopomer-done on colon cancer line: HCT116 and is a very
aggressive cancer line in culture
2) Colon Cancer Cells (watched administer drugs on 7/11/13)
• Ex: breast cancer cells deprive glucose in culture die
Metastatic breast cancer cells in brain deprive glucose in culture they can
still proliferate
•
Study in the 30’s where grad students ate food that were deprived a
certain amino acid and they recorded who got sick
•
In cancer cells, if a drug isn’t working, it’s because the cancer cells is
literally pumping out the drug
•
Zheng is presenting his data at the Summer Institute Life of Sciences
(SILS) research symposium at Weill 224 from 5:18-5:30 on August 5
Zheng Ser-Undergraduate Student
Researcher
Day 31 with Sam-Monday
8/5/13
9:00-11:30- Research on Mcf10a cells
• The MCF 10A cell line is a non-tumorigenic epithelial cell line. They are
from a female Caucasian of 36 years who had fibrocystic disease.
Symptoms include noncancerous breast lumps.
• The cells are positive for epithelial sialomucins, cytokeratins and milk
fat globule antigen.
• They exhibit three dimensional growth in collagen, and form domes in
confluent cultures.
• Thus far, the cells have shown no signs of terminal differentiation or
senescence.
• The line is responsive to insulin, glucocorticoids, cholera enterotoxin,
•
•
•
•
•
•
and epidermal growth factor (EGF).
By electron microscopy the cells display characteristics of luminal
ductal cells but not of myoepithelial cells.
They also express breast specific antigens as detected by positive
reaction with MFA-Breast and MC-5 monoclonal antibodies.
The calcium content of the medium exerts a strong effect on the
morphology of the cells.
Research on HCT116 cells
Colorectal carcinoma from an adult male
This line has a mutation in codon 13 of the ras proto-oncogene, and
can be used as a positive control for PCR assays of mutation in this
codon.
Adherent culture properties
12:00-12:45-Heated up 6% Lab Agar in microwave, cleaned up the hood
with 70% ethanol, added 2 mL of the agar to 18 mL of the media, added
2 mL to each well in a 6 well petri dish, covered the 6 well petri dish in
saran wrap and put it in the 4°C refrigerator for 1 hour.
2:00-Agar and media in 6 well plates are completely solid; putting them
in the incubator to thaw so that we can add the cells in about an hour
2:40- Heated up Seaplaque agar in Stipanuk Lab’s microwave (2 min+2x
30 sec)
3:00-3:45-Looked at HCT116 cells under microscope, and about 60%
confluency. Trypsinized cells (remove old media, add sterile PBS, remove
PBS, 1.5 mL of trypsin, wait 5 mins, 1 mL to collect and scrape cells,
another mL to make sure you get cells, centrifuge). Micropipetted 75 μl
sample into moxi Z cassette.
http://www.orflo.com/overview.html
Touch screen to start and the measurement takes 8 seconds, no need for
reagents or dyes.
With 2 mL of media, there were 1.93x10^6 cells/mL. With added 3 mL of
media (5mL) there were 1.64x10^6 cells/mL [Test 245].
(1640000 cells/mL)(x)=(16mL)(10,000 cells/mL)
=0.9756098, 97.5609756 μl ≈ 100 μl
Lastly, put 6 well petri dish in 4°C refrigerator.
Day 32 with Sam-Tuesday
8/6/13
Folate (folic acid) plays a vital role in DNA synthesis, amino acid metabolism,
and the generation of methyl groups.
10:30-11:30-(DOX/PHGDH experiment) Added water to the polar
metabolites that we extracted 7/2/13 [LIKE DISSOLVES LIKE]. Mixed
eppendorf tubes on the vortex mixer and then put them in centrifuge for 10
mins. Then pipetted out the metabolites and put them in newly labeled
containers then set them up in the Mass Spec.
1:30- Microwave has arrived. Please note: nothing can be seen with the
cells for the soft agar assay. Prof. Locasale said that you won’t really be able
to see changes until a few days and when you do they grow in colonies (not
to be mistaken for contamination of bacteria cells or something).
2:30-4:10-Trypsinize Mcf10a cells (remove media, add PBS, remove PBS,
and add trypsin) under hood and then split them. Collect cells with a
micropipette, put the 15 mL tube in the centrifuge for 5 minutes, and
remove all media but the pellet. Then re-suspend with 9 mL of media, add 9
mL of media to each plate, and 1 mL of cells to each plate. “Mcf10a P2
8/6/13 CM”
Day 33 with Sam-Wednesday
8/7/13
9:00-Looked at my Mcf10a cells
split yesterday 8/6/13 P2, under
the microscope and they are
about 15% confluent.
Day 34 with Sam-Thursday
8/8/13
9:30-Looked at my Mcf10a cells split 8/6/13 P2,
under the microscope and they are about
25% confluent.
2:00-Cells are contaminated in 6-well petri
plate with soft agar
3:30-(+/- DOX experiment) Watched Sam
under the hood remove old media from her
cells, add cold PBS, then flash freeze the
plates in liquid nitrogen and then bring
downstairs to -80°C freezer
4:00-6-well plate with +/- DOX, We scraped
off cells with a cell scraper, micropipetted
them into eppendorf tubes, and then put them
in the centrifuge at max speed for 15 minutes.
4:45-Sam
micropipetted the
metabolites and
methanol into newly
labeled eppendorf
tubes and then put them in the speed vac
concentrator for 3 hours since there is about 500
mL of methanol.
4:50-Heated up Seaplaque in microwave 2 mins
+ 2x30 sec and is now cooling down. We are
going to plate the cells in about 10 minutes.
5:00-5:50-Trypsinizing the cells but messed up. Note, do
not remove trypsin with HCT116 cells. So I plated HCT116
cells on a 10 cm petri plate and we’ll have to wait a couple
days.
Day 35 with Sam-Friday
8/9/13
9:30-Looked at my Mcf10a cells under the microscope P2 split 8/6/13 and
they are 100% confluent and should be split today. The HCT116 cells split
yesterday are about 15% confluent.
Mcf10a cells
HCT116 cells
11:30-Prepared the lysis buffer and thawing +/- DOX cells from -80°C
freezer
11:45-Scraped cells, pipetted them into new eppendorf tubes, and let it sit
for 10 mins. Then put in the centrifuge for 30 minutes.
12:30-Pipetted protein-supernatant (liquid only) into new eppendorf tubes
then put in the 4°C fridge.
12:45-Put media in the water bath, going to split cells in about 15 minutes.
2:00-Lab Meeting Today-Mahya is presenting:
“Characterization of one-carbon metabolism in cancer”
4:00-Split cells-trypsinize cells for 30 minutes then plate them
Day 36 with Sam-Monday
8/12/13
9:30-Looked at my cells under the microscope, the HCT116 cells are 95%
confluent and the Mcf10a cells are 95% confluent.
Rinsed the protein with TBST: 1x at 1:30, 2x at 1:49, 3x at 2:01, and then
at 2:20 added primary antibody for 1 hour
3:30-Trypsinized Mcf10a cells
Again rinsed the protein with TBST: 1x at 3:30, 2x at 3:45, 3x at 4:00
4:30-Plated 7 petri dishes with Mcf10a cells: 3 with +DOX Day 1, 2, 3 and 3
with -DOX Day 1, 2, 3 and then one with normal media.
4:45-Imaged Sam’s protein anti-PHGDH +/- DOX with the Chemidot at the
next door lab
Using the Moxi Z, there were
84,000 cells/mL, and we added 1.2
mL to each new petri dish so that
100,800 cells were added to each
plate.
Day 37 with Sam-Tuesday
8/13/13
4:45-Looked at –DOX Day 1 plate under the microscope, and it was
crowded in the middle but not around the edges so it probably wasn’t mixed
and distributed well. The +DOX Day 1 plate was more evenly distributed.
About 70% confluency.
4:50-Trypsinized Day 1 +/- DOX Mcf10a cells (approximately 24 hours
from when they were plated)
5:25-Counted cells with the Moxi Z cell counter
Day 38 with Sam-Wednesday
8/14/13
11:15-12-Went to a Fisher Products Sale at Baker Lab with Carlie and got a
periodic table of elements poster and other things 
4:45-Trypsinized cells
5:25-Counted +/- DOX
Mcf10a cells Day 2 with the
Moxi Z
Day 39 with Sam-Thursday
8/15/13
4:45-Looked at cells under the microscope (90-95% confluent) and then
trypsinized them
5:25-6:00-Counted +/- DOX Mcf10a cells Day 3 with the Moxi Z
DATE
Plated
+DOX
-DOX
Cells/mL
mL
Cell count
Avg Cell
Width (μm)
Cells/mL
mL
Cell count
Avg Cell Width
(μm)
84,000
(Test 252)
1.2
100,800
5.065
84,000
(Test 252)
1.2
100,800
5.065
239,000
(Test 255)
5
1,195,000
17.432
268,000
(Test 254)
5
1,340,000
17.42
815,000
(Test 258)
5
4,075,000
13.575
874,000
(Test 257)
5
4,370,000
13.575
627,000
(Test 260)
14
8,778,000
14.616
611,000
(Test 259)
14
8,554,000
15.041
8/12/13
Day 1
8/13/13
Day 2
8/14/13
Day 3
8/15/13
3a
3c
Last Day 40 with Sam-Friday
8/16/13
2:00-Last Lab Meeting Today-Sam and I are presenting
3d