Nov 01, 2014

Public workspaceBlasticidin S HCl in Mammalian Culture Determine Working Concentration

DOI
dx.doi.org/10.17504/protocols.io.cikucv
Sean Seaver
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DOI: dx.doi.org/10.17504/protocols.io.cikucv
External link: http://store.p212121.com/blasticidin-s-hcl/
Protocol Citation: Sean Seaver: Blasticidin S HCl in Mammalian Culture Determine Working Concentration. protocols.io https://dx.doi.org/10.17504/protocols.io.cikucv
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
Created: October 17, 2014
Last Modified: March 27, 2018
Protocol Integer ID: 300
Abstract
The working concentration of blasticidin S HCl for use in mammalian cell gene selection can range anywhere from 2-100μg/mL depending on the cell lines used.

Optimal doses typically range from 2-20μg/mL

However, we recommend performing a kill curve to determine the optimal dose for your cell lines.
Materials
STEP MATERIALS
ReagentBlasticidin S HClP212121Catalog #TE-B001
ReagentBlasticidin S HClP212121Catalog #TE-B001
ReagentBlasticidin S HClP212121Catalog #TE-B001
ReagentBlasticidin S HClP212121Catalog #TE-B001
ReagentBlasticidin S HClP212121Catalog #TE-B001
ReagentBlasticidin S HClP212121Catalog #TE-B001
ReagentBlasticidin S HClP212121Catalog #TE-B001
ReagentBlasticidin S HClP212121Catalog #TE-B001
Protocol materials
ReagentBlasticidin S HClP212121Catalog #TE-B001
ReagentBlasticidin S HClP212121Catalog #TE-B001
ReagentBlasticidin S HClP212121Catalog #TE-B001
ReagentBlasticidin S HClP212121Catalog #TE-B001
ReagentBlasticidin S HClP212121Catalog #TE-B001
ReagentBlasticidin S HClP212121Catalog #TE-B001
ReagentBlasticidin S HClP212121Catalog #TE-B001
ReagentBlasticidin S HClP212121Catalog #TE-B001
ReagentBlasticidin S HClP212121Catalog #TE-B001
ReagentBlasticidin S HClP212121Catalog #TE-B001
ReagentBlasticidin S HClP212121Catalog #TE-B001
ReagentBlasticidin S HClP212121Catalog #TE-B001
Plate 5x10^4 blasticidin sensitive host cells in 6-12 wells of a 24-well plate using appropriate media (without blasticidin) and incubate overnight.
Duration24:00:00
ReagentBlasticidin S HClP212121Catalog #TE-B001
Replace media with fresh media containing 6-12 different concentrations (one for each well) of blasticidin S HCl.
Duration72:00:00
ReagentBlasticidin S HClP212121Catalog #TE-B001
Continue replacing media with fresh blasticidin S HCl containing media every 3-4 days and monitor cells for normal activity.
Duration72:00:00
ReagentBlasticidin S HClP212121Catalog #TE-B001
After 5-7 days, determine the lowest concentration of blasticidin S HCl containing media that killed 100% of blasticidin sensitive host cells.

This is the optimal dose to use for your selection procedure.
Duration144:00:00
ReagentBlasticidin S HClP212121Catalog #TE-B001