TITLE: Puromycin titration of cancer cell lines
AUTHORS: Emily Souster, Verity Goodwin, Adam Jackson, Charlotte Beaver, Rizwan Ansari, Fiona Behan, Mathew Garnett

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to identify the most suitable puromycin concentration for the selection of Cas9 positive cancer cell lines transduced with guideRNA library virus.</div><div class = "text-block">Process diagram:</div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/b94e6458af3e4aa17dc6c162a5d28b87f529d83e9d05c0764f6eb3fffa72f21d/cchjbdsax.png" /></div></div>

[STEPS]
?. [Day 1: Titration plate set up]
Detach, collect and count cells by following Steps 1-8 of the protocol: Passaging adherent cancer cell lines.
?. [Day 1: Titration plate set up]
Resuspend 1x106 cells in media, at a concentration of 2x105 cells/ml.
5 mL
?. [Day 1: Titration plate set up]
Using a 1mg/ml stock of puromycin, prepare five dilutions at 2x final concentration , by diluting the stock in media as shown in Table 1, column C & D. (When the 2x antibiotic concentration is diluted with an equal volume of cell suspension it will result in the final concentration shown in Table 1, column B).   ABCDE1  2 x
  concentration (µg/ml)  Final
  concentration (µg/ml)1mg/ml stock
  puromycin (µl)Media (ml)Total (ml)2  2    1    10    4.99    5.0  3  4    2    20    4.98    5.0  4  6    3    30    4.97    5.0  5  8    4    40    4.96    5.0  6  10    5    50    4.95    5.0
Prepare a minimum of 5ml of each 2x antibiotic so that the volume is adequate for loading a multi-channel pipette without bubbles.Antibiotic dilutions should be prepared fresh on the day that they are required.
ABCDE1  2 x
  concentration (µg/ml)  Final
  concentration (µg/ml)1mg/ml stock
  puromycin (µl)Media (ml)Total (ml)2  2    1    10    4.99    5.0  3  4    2    20    4.98    5.0  4  6    3    30    4.97    5.0  5  8    4    40    4.96    5.0  6  10    5    50    4.95    5.0
Puromycin is toxic if swallowed and harmful in contact with skin.
?. [Day 1: Titration plate set up]
Pipette  cell suspension to the first 3 wells of rows B-G in a 96-well plate (row A is used a control with no cells, to subtract background luminescence).
75 µl
Always seed 3 wells per row as the titration is carried out in triplicate. Therefore, a 96-well plate can be used to titrate up to 4 cell lines at a time (see Fig. 1).
?. [Day 1: Titration plate set up]
Pipette  media to the first 3 wells of row A, and  media to the first 3 wells of row B in a 96-well plate.
150 µl
75 µl
?. [Day 1: Titration plate set up]
Pipette  of the puromycin 2x concentrations into the first 3 wells of rows C-G, to achieve the final concentrations as per the plate layout shown in Fig. 1.
75 µl
?. [Day 1: Titration plate set up]
Incubate at  , 5% CO2 for approximately 72 hours.
37 °C
We use a testing time of 72 hours, but depending on the cell line, different time scales such as 24 or 48 hours could be used to pick the highest concentration. Or for particularly resistance cell lines, the testing time can be extended to reflect the amount of time the cells will be in selection.
?. [Day 4: Assessing cell viability using CellTiter-Glo ]
Thaw CellTiter-Glo 2.0 reagent and equilibrate to room-temperature prior to use. Mix by gently swirling to obtain a homogeneous solution.
The CellTiter-Glo reagent can be stored at   and is stable for up to 4 freeze-thaws; thawed reagent can be kept at   for up to 5 months.CellTiter-Glo is light sensitive so should be stored in tin foil, and used in a cell culture hood with the light off where possible.
?. [Day 4: Assessing cell viability using CellTiter-Glo ]
Remove the 96-well plate from the incubator and allow to equilibrate to room-temperature for 15 minutes.
?. [Day 4: Assessing cell viability using CellTiter-Glo ]
Using a multi-channel pipette, add  CellTiter-Glo reagent to each well (1:6 dilution) and mix gently by rocking the plate back and forth. Incubate at room-temperature for 10 mintues (wrap plate in blue roll/foil or keep away from light where possible).
25 µl
?. [Day 4: Assessing cell viability using CellTiter-Glo ]
Use an appropriate plate reader to record the luminescence of each well.
The plate reader should be set to an integration time of 1 second per well, and optimised for a peak emission wavelength of 560nm.
?. [Day 4: Assessing cell viability using CellTiter-Glo ]
Create a kill curve as follows:- Average the triplicate luminescence values to get a single value for each condition.- Subtract the average background luminescence (row A, media only) from the other averaged vaues.- Divide the average luminescence for 1, 2, 3, 4 and 5µg/ml by the 0µg/ml average to get a relative percentage viability.- Plot these values on a graph to create a kill curve.The 'kill concentration' is the lowest concentration of puromycin which results in cell dealth of approximately 100% after 72 hours + 1µg/ml to account for difference in scale up. For example, the 'kill concentration in Fig. 2 is 3µg/ml.