2019 F

Submitted by kdorfman on Thu, 09/12/2019 - 15:46
date prep
9/19 triple stained LLCPk slides: DAPI, rat anti-alpha tubulin & anti-rat-FITC, red phalloidin
9/26 thaw LL flask
9/27 change medium
9/28 split into large flask
9/30 put 5 x 10^5 cells into each of 9 flasks
10/1 students nucleofect
10/3 Start G418 selection of transfected LLs
10/9 Thaw 3t3 into DMEM, spin down, resuspend, plate on coverslips. No adherence (or all dead?) Make new DMEM, replace medium in flasks
10/10 Stop selection - cells look bad. Replace selection medium with regular F10Hams
10/11 very few adherent cells.
10/15 Thaw LL-GFP-alphas,HeLa-H2B-GFP, Mcherry alpha tubulin
(get HeLa GFP alpha tubulin from Pat)
3t3s look fine
split transfected cells into one 25 cm2 flask to grow up, one 12.5 cm2 flask to continue selection
10/16 Plate in coverslip dishes for lab on 10/17 (had to be done in Pat's lab)
10/17 live cells for mitosis (made in Pat's lab: HeLa-GFP-tubulin, LLCPk-GFP tubulin)
thaw LLCPk-GFP-actin for use in motility lab 10/22
thaw LLCPk-GFP-tubulin to test freeze/thaw protocol
split 3t3s to grow up for live cell observations 10/22
split LL parentals in case anyone wants to repeat nucleofection
split nucleofected cells for students; they take over care of them 10/24
10/21 plate (coverslip bottom dishes) 3t3, LL-GFP-actin from flasks
make big flask of LL-GFP-actin to freeze
thaw b16, spin down, resuspend in DMEM, plate (coverslip dishes)
froze 3 vials HeLa-GFP-H2b/mcherry-tubulin
10/22 Bioimaging students split their 2 flasks of nucleofected cells (G418/unselected); made 2 new flasks, 2 coverslip dishes.
10/23 Set up sterile hoods with all equipment students will need.
Freeze 3 vials of 3t3s
10/24 students split nucleofected cells
10/25 split LL-GFP-tub, LL-GFP-actin and freeze 2 vials each
split HeLa-GFP-tub: very strange - some cells never balled up.
10/28 split LL-GFP-tub: 1 flask with 500,000 for nucleofection 10/29, one to keep culture going
split better looking parentals just to keep going
HeLas look OK, but sparse
10/29 students split cells, work on projects
10/30 make 1 flask of LL parentals to nucleofect, 1 flask of LL-GFP-tubulin (plus 1 flask each to perpetuate)
10/31 students work on their cells
11/01 inner incubator door was open, temp was 28C, CO2 was 3
replace medium on HeLas (cultures are pretty sparse)
replace medium on parentals
split LL-GFP-actin to 12.5 flask
11/04 plate 2 coverslip dishes of parental LL's for ER Tracker treatment
make 2 12.5 flasks for propagation
11/06 split LL-GFP-tubulin: 2 full flasks for nucleofection, one for propagation
aliquot 40 mL trypsin
freeze 3 vials HeLa-GFP-tubulin
one flask HeLa-GFP-tubulin in case students want it
split flask of LL-GFP-actin in case students want it
11/08 Kate sick
11/10 split parentals
split LL-GFP-actin
refeed HeLa-GFP-tubulin
aliquot new F10-Hams & DMEM
11/11 Split BL & AM nucleofected cells: 1 flask for nucleofection, one for propagation
G418 selection @ 2 mg/mL
11/12 students work on cultures
11/13 plate LL-GFP tubulin for nucleofection on 11/14 (with students)
11/14 pat brings over IF LL's (intermediate filament eGFP vimentin)
11/15 check IF flasks: all light. Split on Monday
split LLCPK parentals
split HeLa-GFP-tubulin
11/18 8:30 am help students (AZ) count to prep cells (LL-IF-GFP) for nucleofection
split IF flasks for freezing (2 75cm2 flasks), student projects (2 25 cm2 flasks- each with 500,000 cells in case anyone wants to nucleofect them on Tuesday)
11/19 split LL-GFP-tubulin: 1 heavy, 1 light
11/20 split heavy LL-GFP-tubulin, count, put 500,000 in flask for Sam & Brian to nucleofect
11/21 students work
11/22 LLCPK GFP-IF: freeze 3 (2?) vials from T75 flask; split into T25 flask "in case"
LLCPK GFP-tubulin: split light
J & G: split; will split again 11/29 to make a nucleofection flask 12/1
LLCPK Lifact Actin 488 split into T75 flask for freezing; small flask "in case"
KTRM: change medium in dish and flask
LLCPK parentals: split lightly "in case"
HeLa GFP-Tubulin split lightly "in case"
11/25 ASBK split 2 flasks
GOJC H2B: split
Z&A: actin split 1 flask + G418,
Z&A IF change medium plut G418
SLBM split GFP-Tub 2
AMBL IF+Actin split plus G418 reduced by half
SACW will come in to do their own
11/27 Freeze LifAct-488 LL
SLBM split "s1" (or wait till 11/29?)
SLBM split s2 flask LL-red-mitochondria to a 75 flask to freeze Friday
11/29 LLGFP-GFP-tub split (so students can split on 12/1 for nucleofection 12/2)
SLBM freeze T75 (get labels from Sam & Brian)
HeLas look OK, but sparse

Frozen cell lines