Cell Culture


Maintenance Log for U251

August 5, 2016
P1->P2
Was seeded into two flasks
“high” seeding density is 272,000/3=90,666.6667 cells/mL
“low” seeding density is 272,000/6=45,333.3333 cells/mL
2 flasks of P1 U251 left
2 flasks of P2 U251 there

Monitor how much they grow for well seeding

August 8, 2016
U87
Seed high is at 30% confluency
Seed low at 18% confluency
  P1s were at 90 and 75, passaging both
75 660000 cells/ml seeding 0.7 ml
90 880000 cells/mL seedin 0.5 mL
Currently there are 4 flasks of U251

August 9, 2016
P2 seed low did not look good, threw it out
P2 seed high has 20% confluency, media changed
P2 from 75 has 20% confluency, media changed
P2 from 90 has 25% confluency, media changed

August 10, 2016
P2 seed high, 30% confluency, decide tomorrow, should probably passage Friday
P2 from 90, 35% confluency, decide tomorrow, should probably passage Friday
P2 from 75, 20% confluency, should probably change media Friday, leave till Monday
Ask dr. Sin about high glucose media for u251, she said U251 grows slower than b. END3 because it has a much lower passage number and is much closer to the primary cells, so as long as cells are growing, it’s fine.

August 11, 2016
P2 seed high, 35% confluency, should monitor tomorrow, if doesn’t grow, add high glucose media
P2 from 90, 35% confluency, monitor tomorrow, but only change with low glucose media
Changing media today to see if cells would grow more with higher glucose (using same media)
P2 from 75, 25% confluency, monitor tomorrow, ask dr. Sin if you can add high glucose media today?
Changing media today to see if cells would grow more with higher glucose (using same media)

August 12, 2016
P2 seed high, 40% confluency, change media
P2 from 90, 45% confluency, change media
P2 from 75, 30% confluency, change media today

August 15, 2016
P2 seed high, 65% confluency, change media
P2 from 90, 75% confluency, passaged 0.5 of total cells into flask 1, other half in flask 2
P2 from 75, 40% confluency, throwing away

August 16, 2016
P3 flask 1, 50% confluency, seeding higher worked as expected confluency was 35%, from now in seed in at least 30%, healthy adherence is observed
P3 flask 2, 50% confluency, healthy adherence is observed
P2 seed high, 65% confluency, using for mini experiments, cells are probably not very healthy, because they have not grown since yesterday and have been in the same flask for 10 days but they are good for a trial run

August 17, 2016
P3 flask 1 &2, 65% confluency, changing media

August 18, 2016
P3 flask 2, 80% confluency, cells look healthy and are growing!
passaged cells (~30% of cells resuspended in 3mL of media) into P4 flask 2

P3 flask 1, around 30% confluency, left to grow

August 19, 2016
P4 flask 2, 35% confluency, changing media
P3 flask 1, 80% confluency, passaging into 1, 3/8th of total cells were passaged into flask 1
240 live cells in 15 squares, the result of this count is much lower than expected, will validate it with how well the number of cells seen on Monday (it will give an idea of seeding density), might be because of aggregates that were seeded into flasks before this count sample was taken

August 22, 2016
Both flask 1 & 2 are at 70% confluency, changing media for both, tomorrow using one to seed cells in 24 well plate and the other to passage into 3 flasks

August 23, 2016
Both are around 80, passaging each one into new flasks today, also using both to seed 24 well plate

August 24, 2016
Flask 1 is at 40 and flask 2 is at 45, healthy adherence us observed

August 25, 2016
Both flasks are at around 70%, changing media for both flasks today.

August 26, 2016
Flask 1 is at 75% confluency, passaging to 37.5% (1-1) and 37.5% (1-2)
Flask 2 is 85% confluency, passaging to two 42.5q% flasks

August 29, 2016
Flask 1-2 80% confluency used for exp B
Flask 1-1 85% confluency used for exp B
Flask 2-1 70% confluency media changed
Flask 2-2 75% confluency used for exp B

August 30, 2016
Flask 2-1 passaged, 0.5 of total cells were transferred into each flask (2 flasks total)

September 1, 2016
Passage 7 flasks 1 and 2 are both around 70% confluent, media changed

September 2, 2016
Flask 1 is still around 70% confluent but lots of dead cells and debris floating around - just changed media
Flask 2 is 90% confluent, looks very good and healthy, passaging into flask 1 and flask 2 (p8)

September 5, 2016
Flask 1 p7 from Aug 30th, still at 70% confluency, throwing it out
Flask 1 p8, 85% confluency, passaging into two today
Flask 2 p7, 65% confluency, changing media, hoping to freeze tomorrow

September 6, 2016
Both flasks at 50%
Flask 2 p8 90% confluency, freezing into two vials today

September 7, 2016
Flask 1 is at 70%, flask 2 is at 60%, changing media for both

September 8, 2016
Flask 1 is at 80% and flask 2 is at 75, passaging flask 1 and leaving flask 2, passaging flask 1 at 30%

September 9, 2016
P10 passaged yesterday is at 50, changing media
P9 at 95, passaging into two

September 12, 2016
P10 1, 85%, seed today for experiment B1 third part
P10 2-1, 65%, change media
P10 2-2, 90%, seed today for experiment B1 third part

September 13, 2016
P10 2-1 95%, passaged today into two

September 14, 2016
Both passages are 65%

September 15, 2016
Flask 2 is at 85% passaging today into two
Flask 1 is at 75%, changing media

September 18, 2016
Both flasks 90% confluency, passaging flask 2, 1/3 flasks 1, 1/5 flask 2

September 19, 2016
Flask 1 35%
Flask 2 40%

September 21, 2016
Flask 1 60%
Flask 2 75%
Changing media for both

September 23, 2016
Both 100% confluent and spreading out over each other
Passaged flask 1 into P15 15%

September 26, 2016
Around 35% confluent
Changed media

September 28, 2016
U251 cells don’t look too good, they haven’t grown since yesterday
Added more media
Hoping they look better tomorrow

September 29, 2016
U251 cells don’t look they have grown, and are contaminated.
Threw out. Need to thaw new cells
Left a flask with media to observe growth of any bacteria

Note: source of contamination may be from Sep 26 when passaging bEND3 cells and working on U251

October 2, 2016
Flask of media looks fine without any observable contamination
Thawed two flasks of P9 cells using following protocol:

  1. Put cells in water bath, while in water bath, label two 15mL tubes and add 4 mL of media to each
  2. Add thawed cells to each tube
  3. Centrifuge
  4. Aspirate media out, resuspend in 3 mL media for each tube and seed T25 with it

October 3, 2016
Flasks 1 and 2 p9 both look good, 20-25% confluency, change media tomorrow

October 4, 2016
Changed media

October 7, 2016
Both flasks around 40, changed media for both

October 10, 2016
Sadly, pooled all the cells and the vaccuum sucked up the pellet. Pelleted down at 1.5 RPM for 4 mins 30 seconds, as opposed to 1 RPM for 5 minutes.
Did not aspirate directly above the pellet, kept a good distance away from the pellet (probably around 4 mL mark out out 6 mL of media in the tube)

Maintenance Log for b.End 3