Haynes Lab:Notebook/Engineering PC-TFs/2015/02/16

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Harvested GAL4EED-Luc Cells from 6-Well Plate
Cells had been incubated with doxycyline for 5 days (imaged day before at the three day dox incubation + 1 day transfection point).

In TC Room.
Vacuumed off old media from each well.
Washed each well with 1mL DPBS.
Trypsinized with 0.5mL Trypsin per well.
Washed and deactivated Trypsin with 3.5mL fresh media per well.
Collected cell samples in correspondingly labeled 15mL conical vial.
Pelleted cells in centrifuge at 1000rpm for 3 minutes.
Aspirated off media leaving approximately 50μL on top of pelleted cells.
Flicked to resuspend cells in 50μL media.
Added 1mL cold FACS to each sample 15mL tube (taken down from 2mL because cell pellet did not look giant).
Placed tubes on ice.
Added 1mL FACS to an empty tube for flow cytometry.
Move everything outside of the TC Room.

Luciferase Assay
Within Gen5.0 select KAH-Luciferase.
Go into Protocol (on left had side of screen) and then double click 'Procedure'.
Double click 'Read'.
Ensure that Luminescence is selected.
Ensure that the Gain is set to 200.
Select the active wells.

Sample Preparation
Assay buffer (stored in -80): 100μL buffer per sample with three repeats per sample. 6 samples * 3repeats/sample = 18repeats -> used 20 repeats in case of pipetting error. 20 repeats * 100μL per repeat = 2000μL buffer required.
Pipetted 2000μL buffer into a 15mL conical tube.
Added 2.5μL/repeat D-luc (stored with buffer in -80). --> 2.5μL/repeat * 20repeats = 50μL D-luc.
Pipetted 50μL D-luc into 15ml conical tube containing the 2000μL buffer. Stored protected from light under box.

Filtered all volume of cells in FACS from each 15mL conical tube experimental sample through strainer caps.
Transfer 3x 100 μL strained cells (three repeats per sample of strained cells) to corresponding 3 wells in 96-well plate (white opaque, flat bottom). (remainder of strained cells on ice for flow cytometry/ counting.)
Loaded plain FACS buffer into three wells as a background control.
Very quickly mixed (by pipetting) 100 uL luciferase activity buffer into each 100 uL cell sample.
Incubated the reaction at room temperature (shielded from light in box) for the 10 minutes.
Used the 96-well plate reader to measure the chemiluminescent signal. Three reads is recommended to capture output at maximum or steady state.

The current recommended program for reading luciferase activity on the Synergy H1 reader is:

  • Protocol: KAH_luciferase
  • Optimized specs:
Double click 'Procedure' to edit
Plate Type: 96 WELL PLATE
Under 'Actions' click 'Read' to add a read step. Check the following:
Detection method: Luminescence
Read type: Endpoint
Integration time 0:01:00 MM:SS:ss
Wavelengths: 1
Gain: 200
Read height: 1.00 mm

Save and export data for analysis. Normalize plate reader values to cell count from flow cytometer (see below).

Flow Cytometry
Open software.
Name the samples in the 96-well plate grid.
Change following settings on the 'Collect' tab.
Under run settings, select run with limited. Choose 20μL volume to count how many cells are in a 20μL volume of your samples.
Have 'fluidics' set to slow.
Pick an empty sample in the 96-well plate format and load a tube of deionized water.
Click 'Run' on the software to clean the machine.
Save the file: make sure to enter the date along with an appropriate name.
If the events are not too plentiful and in a range of interest, then pause the run and discard the data.
Repeat above with empty 1mL tube of FACS.
Load first sample. Gently flick to make sure that no cells are sitting at the bottom of the tube before reading.
Save the data to the selected 96-well plate format cell with corresponding name.
Load the empty FACS buffer after each sample to clean the instrument.
Rinse and repeat until all samples done.
Rinse again with deionized water before turning off the machine.

For data collection, hit the 'histogram' button. You will be making three graphs. The data is stored in the software so an retroactively change and delete the graph types.
The first graph should have side scatter (SSC) on the Y-axis and forward scatter (FSC) on the X-axis.
The second graph should be a histogram with 'count' on the Y-axis and forward scatter on the X-axis.
The final graph should have 'count' on the Y-axis and RFP (FL3-A) on the X-axis.
Click 'zoom out' below the graph to show all data-points. Zoom in as is necessary to produce an appropriate graph of scattering.
Live cells should be densely clustered. Reference online material if unclear.
Line-gate around the cluster of live cells to exclude other particles and dead cells in your data.
If cell count is under 10,000 for this gated data (RECORD THIS VALUE), move to the next row down in the 96-well plate format in the software and title appropriately.
Change the settings from volume (20μL) to count WITHIN GATE. Set this value to 10,000.
Repeat data collection for each sample with FACS washed inbetween.
Export all samples at .FCS and save to external flash drive

Cell Count Data within Gate for 20μL Sample from 2.17.2014

BL01 +Dox: 6630
BL01 -Dox: 4704
BL09 +Dox: 4412
BL09 -Dox: 9075
Negative +Dox: 8453
Negative -Dox: 9815