TITLE: Blasticidin 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 optimum blasticidin concentration for the selection of Cas9 positive cancer cell lines. </div><div class = "text-block">Process diagram: </div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/b94e6458af3e4aa17dc6c162a5d28b87f529d83e9d05c0764f6eb3fffa72f21d/ccg8bdsax.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 5x105 cells in  of culture media, at a concentration of 1x105 cells/ml.
5 mL
?. [Day 1: Titration plate set up]
Using a 10mg/ml stock of blasticidin, prepare four dilutions, at 2x final concentration by diluting the stock in media as show 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 show in Table 1, column B).  ABCDE12 x
  concentration (µg/ml)Final
  concentration (µg/ml)10mg/ml stock
  blasticidin (µl)Media (ml)Total (ml)2  20    10    10    4.99    5  3  50    25    25    4.975    5  4  100    50    50    4.95    5  5  150    75    75    4.825    5
Prepare a minimum of 5ml of each 2x antibiotic to 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.
ABCDE12 x
  concentration (µg/ml)Final
  concentration (µg/ml)10mg/ml stock
  blasticidin (µl)Media (ml)Total (ml)2  20    10    10    4.99    5  3  50    25    25    4.975    5  4  100    50    50    4.95    5  5  150    75    75    4.825    5
A wider range of blasticidin concentrations can be used if necessary, for example 1µg/ml- 200µg/ml, depending on the cell type and the sensitivity of the cell line.
Blasticidin is toxic if swallowed and harmful in contact with skin.
?. [Day 1: Titration plate set up]
Incubate at  , 5% CO2 for approximately 72 hours.
37 °C
?. [Day 1: Titration plate set up]
Using a multi-channel pipette, add  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 blasticidin 2x concetrations into the first 3 wells of rows C-F, to achieve the final concentrations as per the plate layout shown in Fig. 1.
75 µl
?. [Day 1: Titration plate set up]
Using a multi-channel pipette, add  cell suspension to the first 3 wells of rows B-F in a 96-well plate (row A is used as 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 4: Assessing cell viability using CellTiter-Glo at 72 hours]
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 sensitivie 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 at 72 hours]
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 at 72 hours]
Using a multi-channel pipette, add   CellTiter-Glo reagent to each well (1:6 dilution) and mix by gently rocking the plate back and forth. Incubate at room-temperature for 10 minutes (wrap plate in blue roll/foil or keep away from light where possible).
25 µl
?. [Day 4: Assessing cell viability using CellTiter-Glo at 72 hours]
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 at 72 hours]
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 10, 25, 50 and 75µg/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 blasticidin which results in death of approximately 100% of cells after 72 hours. For example, the 'kill concentration' in Fig. 2 is 25µg/ml.