Figure S1.
Optimization of the crosslinking step in the 12× 3D-ExM protocol. (A) An example 3D image of how the lateral and axial expansion factors were determined. The longest length along the major axis and the minor axis of an oval-shaped nucleus were used to determine the lateral expansion factor of the nucleus. (B) An example image of how a surface annotation was created to fit the shape and volume of a nucleus. (C) Left: representative images of HeLa cells treated with or without MNase before and after 4× ExM. Right: quantification of the size of the nucleus of untreated cells or cells treated with 2 U or 16 U MNase using the traditional 4× ExM protocol. (D) Optimization of the concentration of AcX as a crosslinking reagent. Left: representative images of HeLa nuclei when different concentrations of AcX were used for crosslinking in the 12× ExM protocol. Right: quantification of the size of the nucleus of HeLa cells shown on the left. n = 46, 30, 34, 46, 27, 44, and 29 cells (from left to right) pooled from two to three color-coded biological replicates. (E) Optimization of the concentration of GA as a crosslinking reagent. Left: representative images of HeLa nucleus when different concentrations of GA were used for crosslinking in the 12× ExM protocol. Right: quantification of the size of nucleus shown on the left. n = 30, 30, 30, 45, 30 cells (from left to right) pooled from two to three color-coded biological replicates. (F) Optimization of the crosslinking method for the 12× ExM protocol. Left: representative images of HeLa nucleus when cells were incubated with AcX overnight, AcX + GA overnight, GA for 15 min, or GA overnight. Right: quantification of the size of the nucleus shown on the left. n = 30, 30, 31, 31 cells (from left to right) pooled from two color-coded biological replicates. Each dot shown in the quantification plots indicates a single nucleus. DNA was stained with DAPI.

Optimization of the crosslinking step in the 12 × 3D-ExM protocol. (A) An example 3D image of how the lateral and axial expansion factors were determined. The longest length along the major axis and the minor axis of an oval-shaped nucleus were used to determine the lateral expansion factor of the nucleus. (B) An example image of how a surface annotation was created to fit the shape and volume of a nucleus. (C) Left: representative images of HeLa cells treated with or without MNase before and after 4× ExM. Right: quantification of the size of the nucleus of untreated cells or cells treated with 2 U or 16 U MNase using the traditional 4× ExM protocol. (D) Optimization of the concentration of AcX as a crosslinking reagent. Left: representative images of HeLa nuclei when different concentrations of AcX were used for crosslinking in the 12× ExM protocol. Right: quantification of the size of the nucleus of HeLa cells shown on the left. n = 46, 30, 34, 46, 27, 44, and 29 cells (from left to right) pooled from two to three color-coded biological replicates. (E) Optimization of the concentration of GA as a crosslinking reagent. Left: representative images of HeLa nucleus when different concentrations of GA were used for crosslinking in the 12× ExM protocol. Right: quantification of the size of nucleus shown on the left. n = 30, 30, 30, 45, 30 cells (from left to right) pooled from two to three color-coded biological replicates. (F) Optimization of the crosslinking method for the 12× ExM protocol. Left: representative images of HeLa nucleus when cells were incubated with AcX overnight, AcX + GA overnight, GA for 15 min, or GA overnight. Right: quantification of the size of the nucleus shown on the left. n = 30, 30, 31, 31 cells (from left to right) pooled from two color-coded biological replicates. Each dot shown in the quantification plots indicates a single nucleus. DNA was stained with DAPI.

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