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. 2016 Feb 11;164(4):722–734. doi: 10.1016/j.cell.2015.12.054

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© 2016 The Authors

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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Figure S6 — Cholesterol Estimation on Phagosomes, Related to Figures 4 and 5

(A) Known cholesterol standards were prepared by dilution. Two sets of identical dilutions (Series1, Series2) were used to determine OD at 570nm. The average OD₅₇₀ at a particular cholesterol dilution was determined from these two series. The EP and LP samples (having equal PC) are also shown on the right. The difference in color (i.e., difference in cholesterol) between EPs and LPs is obvious from this image. The EP and LP images have a darker background compared to the cholesterol standards because the camera flash did not illuminate all samples uniformly when this picture was taken. This has no bearing on the measurements of individual wells done in a plate reader. See section 12 of Supplementary Methods for more details.

(B) The average of OD₅₇₀ (Series1+Series2)/2 is plotted as a function of the (known) cholesterol content of these samples. A linear increase in OD₅₇₀ can be seen with increasing cholesterol. The red line is a linear fit. The OD₅₇₀ measured for EPs and LPs (red circles) corresponds to amounts of cholesterol mentioned on the X-axis. The ratio of cholesterol on LPs:EPs is also calculated ( = 1.79; top inset).

(C) EP and LP samples were subjected to LC/MS/MS to measure free cholesterol and PC. Measurements were done at Avanti Polar Lipids. The amount of cholesterol (after normalizing for PC) is plotted. The ratio of cholesterol (LPs:EPs) is also calculated.

(D) EP and LP samples (having equal PC) were subjected to GC-MS to measure the amount of free cholesterol. The peak for cholesterol is shown. Calculated area under the peak is mentioned.

(E) A calibration curve was prepared by GC-MS using known cholesterol standards. The amount of cholesterol in EP and LP samples was calculated using this calibration curve (calculated amounts mentioned in figure). The LP:EP cholesterol ratio is also calculated (top). Note that the calibration curve is linear and values of cholesterol on EPs and LPs fall within the linear range.