20. Leave a small volume of medium (~50 μL) in the microcentrifuge tube after every wash, as this minimizes the incidence of
aspirating out the loose cell pellet formed by the small number
of cells.
21. Wash the cover glass with enough PBS to remove unbound
fibronectin. This step improves cell attachment as unbound
fibronectin in solution can saturate the binding sites on the
cells.
22. Detailed methods for identifying telomere loci and analyzing
colocalization between FISH and MB signals in three dimensions can be found in [27].
23. Colocalization is not perfect because of the non-consensus
nature of the α-satellite sequence within centromeres [16, 22].
24. Parameters used in this experiment are determined based on
our dataset and experience. The operators should determine
the optimal parameters and filter settings for their own
experiments.
25. Although frame interval in cellSens Dimension for time-lapse
acquisition was theoretically set to 100 ms, the real frame
interval was ~104 ms due to the downtime between
acquisitions.
26. Linking max distance defines the maximal displacement
between two particles in adjacent frames that are considered
to belong to the same track. Gap-closing max frame gap determines the maximal frame interval allowed between particle
observations that may be considered as the same particle.
Gap-closing max distance decides the maximal displacement
allowed between particle observations with the gap-closing
frame interval that may be considered to belong to the same
track. For different datasets, these parameters could be altered
accordingly.
27. For calculating the mean square displacement (MSD), tracks
containing at least 15 time lags (Δτ) were selected. The
2-dimensional diffusion coefficient (D eff ) was calculated by
linear regression of the first 25% of total time lags of the
MSD vs. Δτ dataset with minimum fitting threshold of
R
2
¼ 0.8.
Acknowledgments
This work was supported by grants from the National Key R&D
Program of China (2016YFA0501603), the National Natural Science Foundation of China (Nos. 31771583 and 81371613), the
Beijing Natural Science Foundation (7162114), and China’s 1000
Young Talent Award program.
370
Xiaotian Wu et al.
aspirating out the loose cell pellet formed by the small number
of cells.
21. Wash the cover glass with enough PBS to remove unbound
fibronectin. This step improves cell attachment as unbound
fibronectin in solution can saturate the binding sites on the
cells.
22. Detailed methods for identifying telomere loci and analyzing
colocalization between FISH and MB signals in three dimensions can be found in [27].
23. Colocalization is not perfect because of the non-consensus
nature of the α-satellite sequence within centromeres [16, 22].
24. Parameters used in this experiment are determined based on
our dataset and experience. The operators should determine
the optimal parameters and filter settings for their own
experiments.
25. Although frame interval in cellSens Dimension for time-lapse
acquisition was theoretically set to 100 ms, the real frame
interval was ~104 ms due to the downtime between
acquisitions.
26. Linking max distance defines the maximal displacement
between two particles in adjacent frames that are considered
to belong to the same track. Gap-closing max frame gap determines the maximal frame interval allowed between particle
observations that may be considered as the same particle.
Gap-closing max distance decides the maximal displacement
allowed between particle observations with the gap-closing
frame interval that may be considered to belong to the same
track. For different datasets, these parameters could be altered
accordingly.
27. For calculating the mean square displacement (MSD), tracks
containing at least 15 time lags (Δτ) were selected. The
2-dimensional diffusion coefficient (D eff ) was calculated by
linear regression of the first 25% of total time lags of the
MSD vs. Δτ dataset with minimum fitting threshold of
R
2
¼ 0.8.
Acknowledgments
This work was supported by grants from the National Key R&D
Program of China (2016YFA0501603), the National Natural Science Foundation of China (Nos. 31771583 and 81371613), the
Beijing Natural Science Foundation (7162114), and China’s 1000
Young Talent Award program.
370
Xiaotian Wu et al.