TITLE: Tissue culture- Purity sorting HEK293T LLP iCasp9 cells
AUTHORS: Raining Wang, Melinda Wheelock

[DESCRIPTION]
This protocol covers the sorting of landing pad cells (HEK293T-LLP-iCasp9-blast) for purity prior to starting VAMPseq experiments. This avoids using cells which have undergone landing pad silencing, and increases the proportion of cells that are recombined during transfection experiments. Cells are sorted on blue fluorescence, as cells which do express the landing pad also express blue fluorescent protein (BFP).

[BEFORE_START]
Ensure that the culture of cells has been growing for several days before purity sorting - do not use freshly thawed cells in this protocol.
Cells should be sorted using an 85-um nozzle for a 4-way sterile purity sort (these are flow cytometer settings).

[STEPS]
SECTION: Preparation of cells
1. Remove media from flask by aspiration.
SECTION: Preparation of cells
2. Wash flask with 10 mL DPBS. Remove DPBS by aspiration.

SECTION: Preparation of cells
3. Add 3 mL trypsin to the culture flask. Place in a 37 °C incubator with 5% CO2 for 5 min.

SECTION: Preparation of cells
4. Visualize trypsinized cells under a microscope to confirm cells have detached.
SECTION: Preparation of cells
5. Add 7 mL D10 media and rinse flask with this media to collect all cells. Transfer this 10 mL of media and cells to a 15-mL conical tube.

SECTION: Preparation of cells
6. Centrifuge at 300 x g, 5 min.

SECTION: Preparation of cells
7. Aspirate off media and resuspend the pellet in 5 mL of flow buffer.

SECTION: Preparation of cells
8. Save 1 mL as a backup in case the sort fails or the sorted cells become contaminated. Centrifuge these cells again, aspirate the media, and resuspend in 1 mL D10 media. Plate in a T-75 flask.

SECTION: Preparation of cells
9. Transfer the remaining 4 mL of resuspended cells (in flow buffer) to two 5-mL round bottom polystyrene test tubes (from here called flow tubes). 

SECTION: Preparation of cells
10. Prepare several flow tubes for recovery of sorted cells - label and fill with 2 mLD10 media.

SECTION: Sort for purity
11. On the flow cytometer software, open your experiment and create a tube within that experiment for the cells.
SECTION: Sort for purity
12. Remove the cap and load the flow tube containing cells into the sample loader. Press “Load” on the software. Analysis should begin automatically, or press “Acquire data”.
SECTION: Sort for purity
13. Gate for single cells by gating in the following order:

FSC-A vs SSC-A to gate for live cells
FSC-A vs FSC-H to gate for single cells in the forward direction
SSC-A vs SSC-H to gate for single cells in the side direction
SECTION: Sort for purity
14. Create a graph that shows BFP fluorescence on the x-axis and choose “histogram” on the y-axis. Adjust the BFP laser setting if needed so that the main peak (with strong BFP fluorescence) is centered over 10∧5.
SECTION: Sort for purity
15. Gate for cells that fluoresce BFP - select a tight area around the positive peak. This will be the sorted population. Note the percentage of the single cell population that expresses BFP. Click on “Record data”. 

If the percentage is >98%, sorting is not necessary.
SECTION: Sort for purity
16. Install the recovery tube in the sorter.
SECTION: Sort for purity
17. In the software’s experiment dashboard, open the “Cytometer settings”, then open a sort layout. 
SECTION: Sort for purity
18. Choose the stall which contains the recovery tube, and select the population to sort into the recovery tube (the BFP-expressing cells gated in step 15). 
SECTION: Sort for purity
19. Click on the “Sort” button in the sort layout window.
SECTION: Sort for purity
20. As the flow cytometer sorts the cells, pay attention to the volumes in both the original flow tube and the recovery tube. Do NOT let the original flow tube run empty, or the recovery tube overflow. 

To stop the sorter, click on “Pause” in the sort layout window - this does not stop the sorter from pulling from the original flow tube. 
Click on “Stop Acquisition” or “Unload” if it is necessary to change the tube on the sample loader. 
SECTION: Sort for purity
21. Sort cells into the prepared recovery tube until the total number of cells needed is reached.
SECTION: Sort for purity
22. Remove tubes from the cytometer and follow instructions for post-sorting cleanup given by the manager of the flow cytometer.
SECTION: Plating sorted cells
23. Centrifuge the recovery tubes at 300 x g, 5 min.

SECTION: Plating sorted cells
24. While the centrifuge is running, prepare a new flask for the desired volume of culture by adding N-1mL to the flask (for a desired 10 mL culture, add 9 mL to the flask now). Add doxycycline and blasticidin to the flask.

SECTION: Plating sorted cells
25. Aspirate the supernatant from the recovery tubes.
SECTION: Plating sorted cells
26. Resuspend the cells in a total of 1 mL D10 media (for 2 recovery tubes add 500uL to each, etc.).

SECTION: Plating sorted cells
27. Add the resuspended cells to the prepared flask.
SECTION: Plating sorted cells
28. Optional: estimate the number of cells by staining the cells with trypan and counting using a hemocytometer.
SECTION: Plating sorted cells
29. Place the flask in a  37 °C incubator with 5% CO2 and check cells until confluence.