07/06/13
- Gal4-EED/luc dox time point (48 hours)
- Gal4-EED/luc PcTF transfection - microscopy & flow cytometry
- SK-N-SH +PcTF & mock - TRIzol prep #1
Luciferase activity assay - time point: 2 days
Cell prep
- Harvested cells (induced on 7/04/13)
- Seeded new 2 new plates with non-induced cells from 7/04/13
- Pelleted all of the dox+ cells, resuspended in 2 mL FACS buffer
Assay reagents
- Used the Biotium Steady-Luc Firefly HTS Assay Kit (same as 6/18/13)
Luc assay
- Filtered 700 μL cells through strainer caps
- Used opaque white Costar plate (from Rege lab)
- Samples loaded in triplicate (by columns)
- Included luc buffer + FACS-buffer "blank" sample (well D1)
- Other steps same as 6/18/13
Cell counts
- Wang lab's Accuri flow cytometer.
- Set machine to read 20 uL of cells
- Be sure to "clean" with water-run in between samples
Sample ID |
Gated count/ 20 μL |
|
Cells/ 100 μL
|
sample 1 |
37,629 |
x5 = |
188,145
|
sample 2 |
34,580 |
x5 = |
172,900
|
sample 3 |
40,096 |
x5 = |
200,480
|
sample 4 |
46,599 |
x5 = |
232,995
|
sample 5 |
46,491 |
x5 = |
232,455
|
sample 6 |
46,254 |
x5 = |
231,270
|
SK-N-SH TRIzol prep
- +PcTF, plate 1
- mock, plate 4
(Cells may need longer to express genes. Previous expt. was done after ~10 days. Will do this prep just in case cell culture declines)
- Used other two plates to passage cells ~1:5 (will prep after returning from SB6.0)
- Cells were 100% confluent
- Discarded growth medium
- Added 2 mL TRIzol directly to plates
- Incubated at r.t. for ~5 min.
- Collected lysed cells from plate with gentle scraping (pipette tip)
- Transferred 500 μL aliquots to 2.0 mL tubes. Stored at -80°C
- 4x PcTF+ samples
- 4x mock samples
|