Jul 23, 2020
Blasticidin titration of cancer cell lines
- Emily Souster1,
- Verity Goodwin1,
- Adam Jackson1,
- Charlotte Beaver1,
- Rizwan Ansari1,
- Fiona Behan1,
- Mathew Garnett1
- 1Wellcome Sanger Institute
- Cellular Generation and Phenotyping
Protocol Citation: Emily Souster, Verity Goodwin, Adam Jackson, Charlotte Beaver, Rizwan Ansari, Fiona Behan, Mathew Garnett 2020. Blasticidin titration of cancer cell lines. protocols.io https://dx.doi.org/10.17504/protocols.io.bgz6jx9e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 01, 2020
Last Modified: July 23, 2020
Protocol Integer ID: 37662
Abstract
This protocol is used to identify the optimum blasticidin concentration for the selection of Cas9 positive cancer cell lines.
Process diagram:
Guidelines
- Ensure the cell suspension is mixed thoroughly to create an even single cell suspension before plating.
- All steps involved in the plate set up, including seeding cells, media, antibiotics and CellTiter-Glo should be carried out using reservoirs and multi-channel pipettes where possible to avoid ergonomic strain and to maintain homogenous solutions throughout.
- It is essential to use black 96-well plates in this protocol, as luminescence can carry over into neighbouring wells in clear plates.
Materials
MATERIALS
CellTiter-Glo(R) 2.0 AssayPromegaCatalog #G9241
Falcon™ 15mL Conical Centrifuge TubesFisher ScientificCatalog #14-959-53A
TrypLE™ Express Enzyme (1X), no phenol redThermo FisherCatalog #12604021
Reagent ReservoirThermo FisherCatalog #9510047
DPBSInvitrogen - Thermo FisherCatalog #14190
10mg/ml BlasticidinInvivoGenCatalog #ant-bl-1
Black walled 96 well plateFisher ScientificCatalog #10419822
Select an appropriate culture media for your cell line. Common culture medias used for cancer cell lines are serum supplemented Advanced DMEM F-12 or RPMI, in the presence of pen-strep.
Equipment
Light Microscope
Microbiology safety cabinet (MSC)
Pipette Boy
Stripettes
Pipettes and tips
Centrifuge
Multichannel Pipette and tips
37 °C , 5%CO2 incubator
Plate reader
Safety warnings
- Blasticidin is toxic if swallowed and harmful in contact with skin.
- CellTiter-Glo is harmful to aquatic life with long lasting effects.
Before start
- Pre-warm culture media to room-temperature.
- Thaw a vial of 10mg/ml Blasticidin.
Day 1: Titration plate set up
Day 1: Titration plate set up
Detach, collect and count cells by following Steps 1-8 of the protocol: Passaging adherent cancer cell lines.
Resuspend 5x105 cells in 5 mL of culture media, at a concentration of 1x105 cells/ml.
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).
Note
- 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.
| 2 x concentration (µg/ml) | Final concentration (µg/ml) | 10mg/ml stock blasticidin (µl) | Media (ml) | Total (ml) | |
| 20 | 10 | 10 | 4.99 | 5 | |
| 50 | 25 | 25 | 4.975 | 5 | |
| 100 | 50 | 50 | 4.95 | 5 | |
| 150 | 75 | 75 | 4.825 | 5 |
Table 1. Preparation of blasticidin concentrations using 10mg/ml stock to achieve a 2x concentration.
Note
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.
Safety information
Blasticidin is toxic if swallowed and harmful in contact with skin.
Using a multi-channel pipette, add 75 µL 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).
Note
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).
Using a multi-channel pipette, add 150 µL media to the first 3 wells of row A, and 75 µL media to the first 3 wells of row B in a 96-well plate.
Pipette 75 µL 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.
Figure 1. Plate layout for blasticidin titration of one cell line.
Incubate at 37 °C , 5% CO2 for approximately 72 hours.
Day 4: Assessing cell viability using CellTiter-Glo at 72 hours
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.
Note
- The CellTiter-Glo reagent can be stored at -20 °C and is stable for up to 4 freeze-thaws; thawed reagent can be kept at 4 °C 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.
Remove the 96-well plate from the incubator and allow to equilibrate to room-temperature for 15 minutes.
Using a multi-channel pipette, add 25 µL 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).
Use an appropriate plate reader to record the luminescence of each well.
Note
The plate reader should be set to an integration time of 1 second per well, and optimised for a peak emission wavelength of 560nm.
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.
Figure 2. Kill curve for a blasticidin titrated cancer cell line.