Z3221319 Photos taken and densities recorded of growing cells

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Cells thawed and plated on 24/06/09

Removed 3 vials from the dewer in the PC2 lab:

PC12T frozen by MV 22/01/98, Tube 7

B35 frozen by MH 3/10/98, Tube 19

RGC5 frozen by MS 29/11/99, Tube 5

Cell densities post-plating were:

PC12 - 70-80 at 10x magnification with clumps

B35 - 100 at 10x magnification

RGC-5 - 100 at 10x magnification

Photos and densities on 25/06/09 - 24 hours post thawing

PC12 - less than 10% survival, because wrong medium was used (DMEM f10 P/S instead of DMEM f5 h10 P/S) - this was changed in an attempt to rescue the remaining cells

PC12 cells 1 day post thawing at 4x magnification. Source: z3221319
PC12 cells 1 day post thawing at 20x magnification. Source: z3221319

B35 - about 50% survived freezedown. Mostly flat cells with neurites

B35 cells 1 day post thawing at 20x magnification. Source: z3221319

RGC-5 - 40-50 at 10x magnification - about 50% survive freezedown.

RGC cells 1 day post thawing at 20x magnification. Source: z3221319

Following replacement of media, photos were taken of each of the 6 plates (2 for each cell type) at each magnification (4x,10x,20x,40x). Therefore, there was a total 24 photos. They are saved on the computer and labelled as <cell line> dish <#> <magnification>. Eg. B35 dish 1 4x

Photos and densities on 29/06/09

PC12 - slowly growing, clumps of living cells starting to appear.

PC12 cells 4 day post thawing at 20x magnification. Source: z3221319

B35 - Confluent, need splitting

B35 cells 4 day post thawing at 20x magnification. Source: z3221319

RGC-5 - Confluent, need splitting

RGC cells 4 day post thawing at 20x magnification. Source: z3221319

Photos were taken of all plates as above.

Photos and densities on 30/06/09

Split Cells

B35 1mL replated - still quite confluent

B35 cells split at 5 day post thawing at 4x magnification. Source: z3221319

B35 0.5mL replated - 75 cells/view at 10x zoom

B35 cells split at 5 day post thawing. 10x magnification. Source: z3221319

RGC 0.5mL replated - 400 round cells/view at 10x zoom, with clumps of flattened cells and ?crystalline structures?

Split RGC cells 5 day post thawing at 10x magnification. Source: z3221319

RGC 0.25mL replated - 200 round cells/view at 10x zoom, with fewer clumps of flattened cells and still some ?crystalline structures?

Split RGC cells 5 day post thawing at 20x magnification. Source: z3221319

Unsplit Cells

PC12 - still growing slowly in clumps

PC12 cells 5 day post thawing at 20x magnification. Source: z3221319

B35 - confluent - will split tomorrow

B35 cells 5 day post thawing at 10x magnification. Source: z3221319

RGC - confluent - will split tomorrow

Cells thawed and plated on 30/06/09

Removed 3 vials from the dewer in the PC2 lab:

PC12T frozen by VC 2/05/06, Tube 4

HT4 frozen by MW 18/07/96, Tube 8

N12 frozen by CS(?) 2/10/01, Tube 1

Cells thawed and plated on 1/07/09

Removed 1 vial from dewer 1 in the G16 lab:

C17-2 frozen by MH 3/10/98, Tube 3

Photos and Densities on 3/07/09

Newer Cells

C17-2 ~ 50 cells at 10x magnification

C17-2 cells 2 days post thawing at 10x magnification. Source: z3221319


C17-2 cells 2 days post thawing at 40x magnification. Source: z3221319

HT4 - confluent on both plates. One plate was split, the other left confluent over the weekend.

HT4 cells 3 days post thawing at 20x magnification. Source: z3221319

N12 - 40-50 rounded cells at 10x magnification. Very few flattened cells.

N12 cells 3 days post thawing at 20x magnification. Source: z3221319

PC12 - 100 rounded cells at 10x zoom

Split Cells

B35 - Both 0.5mL dishes split on the 1/07/09 were 300-400 cells/10x magnification. Both dishes (0.5mL and 1mL) split on 29/06/09, were confluent. Therefore, only 1 dish (from the 1/07/09 pair) was split and the others were discarded.

RGC-5 - Both 1mL dishes split on 1/07/09 exhibited clumps of cells and in each clump, a density of ~ 50 cells/10x magnification. Of the two dishes split on the 29/06/09 (0.25mL and 0.5mL dishes), the 0.25mL dish was still growing in clumps, and the 0.5mL dish was potentially contaminated and was therefore disposed of.

Original Cells

PC12 - both dishes still growing in clumps of rounded cells.

Photos and Densities on 6/07/09

C17-2 - both dishes almost confluent. One dish was split into a 0.5mL and 0.25 mL dish.

C17-2 cells at 20x magnification. Source: z3221319

HT4 - both the unsplit dish (2) and the split dishes (from 3/07/09) were confluent. Split Dish 1 (from 3/07/09) was split again.

HT4 cells at 20x magnification. Source: z3221319

N12 - very few cells at all - don't seem to be growing. Perhaps DMEM f10 media is not ideal.

B35 - All 3 split dishes (from 3/07/09) were close to confluent. Dish 1 was split again.

RGC-5 - The 0.25mL split dish from 29/06/09 is still growing in clumps across plate, as is the split dishes from 1/07/09. The newer dishes are not as dense in the clumps.

PC12 - Original plates from 24/06/09 - very very dense clumps of cells, but not confluent over entire plate. Newer plates from 30/06/09 - about 30 clumps of 5-10 cells at 10x magnification. All cells in both plates are rounded, not flattened.

Original plate of PC12 cells (genetically modified) at 4x magnification. Source: z3221319


Newer plate of PC12 cells (non-genetically modified) at 10x magnification. Source: z3221319

Update on plates as of 7/07/09

HT4 - Split plate 1 from 6/07/09 to two new plates. Therefore, now have the two original plates (3/07/09), plate 2 (6/07/09) and two new plates (7/07/09).

C17-2 - Aplit both 6/07/09 plates. The 0.25mL one to a flask, the 0.5mL one to 2 new dishes. Therefore, now have one original plate (1/07/09), two new plates (7/07/09) and 1 new flask (7/07/09).

B35 - Split both plates from 6/07/09. The 0.25mL one into a flask, the 0.5mL one into 2 plates. Therefore, now have 2 plates (3/07/09), 2 plates (7/07/09) and 1 flask (7/07/09).

PC12 - Both non-genetically modified plates split to one flask. One genetically modified plate (24/06/09) split to two plates. Therefore, now have one original genetically modified plate (24/06/09), two split genetically modified plates (7/07/09) and one non-genetically modified flask (7/07/09).

RGC-5 - Both plates from 1/07/09 split to 4 new plates. Therefore, now have one plate (29/06/09) and four plates (7/07/09).

NT2 (previously called N12 - I think I got the name incorrect) - not growing. May change media to optimem if these cells are needed.

Also made up 2 x 24 well plates (7/07/09). Each plate contains 4 wells of each cell type except for RGC-5, which has 8 wells allocated on each plate. Due to problems splitting the non-genetically modified PC12 cells, the genetically modified cells were used for these 2 24 well plates.

Total: 22 plates, 3 flasks, 2 x 24 well plates

Photos of 24 well plate on 8/07/09, prior to differentiation

HT4

Centre of 24 well plate for HT4 20x magnification. Source: z3221319

C17-2

Centre of 24 well plate for C17-2 20x magnification. Source: z3221319

B35

Centre of 24 well plate for B35 20x magnification. Source: z3221319

PC12

Centre of 24 well plate for PC12 10x magnification. Source: z3221319

RGC-5

Centre of 24 well plate for RGC-5 20x magnification. Source: z3221319

Update on plates as of 9/07/09 and photos of flasks

Following cell culling, growing cells consist of the following:

HT4: 2 plates split on 7/07/09 and 2 flasks split today. No pictures are provided of the flasks as they were just split minutes ago.

C17-2: 2 plates split on 7/07/09 and 1 flask from 7/07/09

C17-2 flask 4x mag. Source: z3221319
C17-2 flask 20x mag. Source: z3221319

B35: 2 plates split on 7/07/09 and 1 flask from 7/07/09

B35 flask 4x mag. Source: z3221319
B35 flask 20x mag. Source: z3221319

PC12: 2 genetically modified plates from 7/07/09 and 1 non-genetically modified flask from 7/07/09

PC12 non-genetically modified flask 4x mag. Source: z3221319
PC12 non-genetically modified flask 4x mag. Source: z3221319

RGC-5: 3 plates from 7/07/09. Will create a flask tomorrow.

NT2: 2 plates from 30/06/09 - still not growing.

Update on cells as of 20/07/09

Tissue culture completely shut down today. All cells frozen down in storage and excess plates disposed of. All flasks harvested for DNA/RNA/Protein extraction.