12. A robust CETSA HT assay requires optimization of the antibody dilution, AlphaScreen reagent dilution, and cell density
to achieve a minimum signal of around 15,000 AlphaScreen
counts.
13. Once an antibody pair has been identified and optimized, the
signal should be tested in the absence and presence of the tool
compound (10 μM) to ensure that compound binding does
not disrupt antibody recognition.
14. When handling cells in PCR tubes or a PCR plate, avoid
touching the bottom of the tubes or wells to maintain consistent temperatures of the cell suspension prior to and after the
heat shock.
15. To ensure compatibility with the Roche LightCycler 480 II,
384-well PCR plates have a particular footprint, which differs
from most standard microtiter plates. The design of these
plates makes them incompatible for certain dispensers. For
use with the Echo 555 acoustic dispenser, an adapter is
required (LabCyte). For use with the Multidrop Combi dispenser, PCR plates must be loaded backward (A1 in the bottom right corner) to ensure correct dispensing into each well.
16. CETSA HT assays typically require cells to be prepared to high
cell densities in the region of 2 Â 10
6 cells/mL to
2 Â 10
7 cells/mL. To avoid cell damage, minimize the time
cells are left at such high densities prior to seeding and ensure
regular mixing to prevent cells settling. High cell densities can
also cause cells to clump together, which can adversely affect
results. To ensure a single-cell suspension, gently draw the
solution of cells up into a syringe and filter through a 40-μm
filter prior to dispensing cells with a liquid dispenser, e.g.,
Multidrop Combi.
17. Following brief centrifugation of PCR plates containing cells
and compound, it is likely that cells will form a pellet at the
bottom of the well. Across several different targets, we have not
observed this to influence the compound responses in CETSA
HT and find that cells do not require resuspension or mixing
prior to, or throughout, the compound incubation.
18. We have found thorough mixing of the cell pellet with the Lysis
Buffer to be essential to observe consistent AlphaScreen
® signal between wells. Consistent signal is also dependent upon
accurate volumes of liquid transfer from the PCR plate to the
ProxiPlate-384 Plus.
214
Joseph Shaw and Christopher Stubbs
to achieve a minimum signal of around 15,000 AlphaScreen
counts.
13. Once an antibody pair has been identified and optimized, the
signal should be tested in the absence and presence of the tool
compound (10 μM) to ensure that compound binding does
not disrupt antibody recognition.
14. When handling cells in PCR tubes or a PCR plate, avoid
touching the bottom of the tubes or wells to maintain consistent temperatures of the cell suspension prior to and after the
heat shock.
15. To ensure compatibility with the Roche LightCycler 480 II,
384-well PCR plates have a particular footprint, which differs
from most standard microtiter plates. The design of these
plates makes them incompatible for certain dispensers. For
use with the Echo 555 acoustic dispenser, an adapter is
required (LabCyte). For use with the Multidrop Combi dispenser, PCR plates must be loaded backward (A1 in the bottom right corner) to ensure correct dispensing into each well.
16. CETSA HT assays typically require cells to be prepared to high
cell densities in the region of 2 Â 10
6 cells/mL to
2 Â 10
7 cells/mL. To avoid cell damage, minimize the time
cells are left at such high densities prior to seeding and ensure
regular mixing to prevent cells settling. High cell densities can
also cause cells to clump together, which can adversely affect
results. To ensure a single-cell suspension, gently draw the
solution of cells up into a syringe and filter through a 40-μm
filter prior to dispensing cells with a liquid dispenser, e.g.,
Multidrop Combi.
17. Following brief centrifugation of PCR plates containing cells
and compound, it is likely that cells will form a pellet at the
bottom of the well. Across several different targets, we have not
observed this to influence the compound responses in CETSA
HT and find that cells do not require resuspension or mixing
prior to, or throughout, the compound incubation.
18. We have found thorough mixing of the cell pellet with the Lysis
Buffer to be essential to observe consistent AlphaScreen
® signal between wells. Consistent signal is also dependent upon
accurate volumes of liquid transfer from the PCR plate to the
ProxiPlate-384 Plus.
214
Joseph Shaw and Christopher Stubbs