SUPPLEMENTARY INFORMATION
Scrutinizing calcium flux oscillations in T-lymphocytes to deduce the strength of stimulus
Susan N. Christo, Kerrilyn R. Diener, Robert E. Nordon, Michael P. Brown, Hans J. Griesser, Krasimir Vasilev, Farid C. Christo and John D. Hayball.
Supplementary Figure Legends
Supplementary Figure S1. Schematic diagram of functionalised material surfaces. Glass slides are coated with a plasma polymer layer (PPL) to allow covalent attachment of streptavidin protein. Surfaces are blocked with a bovine serum albumin solution prior to the incubation of biotinylated ligands (biotin-CD3(Fab) shown here). Primary naïve T cells loaded with the calcium dyes Fluo-4AM and Fura-Red AM are deposited onto the funtionalised surfaces and fluorescence time lapse images of calcium flux recorded using confocal microscopy.
Supplementary Figure S2. Calcium oscillations quantified with a wavelet transformation using a Haar basis function. (a) A wavelet scale factor of 40 was selected to reject high frequency noise and low frequency baseline variations. (b) Local minima of the transformed data corresponded to the upstroke of calcium spikes (green trace), whereby a threshold crossing value of -0.5 (blue trace) was used to identify the time intervals to find local minima for individual responding cell graphs (threshold crossing; red arrow).
Supplementary Figure S3. ECDF plots of time to first oscillation for total population showingindividual comparisons against anti-CD3 (blue, solid line) and anti-CD3(Fab) (blue, dotted line). Individual comparisons were plotted for anti-CD3+anti-CD28(adh) (black, solid line); anti-CD3+anti-CD28(sens) (thick black, solid line); anti-28(adh) (red, solid line); anti-CD28(sens) (thick red, solid line); anti-CD3(Fab)+anti-CD28(adh) (black, dotted line); and, anti-CD3(Fab)+anti-CD28(sens) (thick black, dotted line). Cox regression analysis was used to determine p-values between the individual comparisons.
Supplementary Figure S4.ECDF plots of time to first oscillation for total and responding population between anti-CD3(Fab)+anti-CD28(sens) (thick black, dotted line) and anti-CD28(sens) (thick red, solid line).Cox regression analysis was used to determine p-values between the individual comparisons.
Supplementary Figures
Supplementary Figure S1
Supplementary Figure S2
Supplementary Figure S3
Supplementary Figure S4
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Supplementary Table S1. Exclusion criteria for manual analysis of single cells
Exclusions and Requirements / ReasoningCells that are in focus at the first recorded interval (t= 1 sec) are excluded ; this also applies to cells that travel into the field of view in focus at any time throughout the recording period / Analysis of the time to first oscillation are skewed if the exact time of adhesion cannot be observed
After adhesion, cells must remain in the field of view throughout the entire recording period; in focus cells that move out of the field of view, even if they calcium flux, are excluded / Detecting cells that move out of the field of view will give an incomplete fluorescence analysis of the given period in which the cell is in focus
At any time after a cell of interest comes into focus, it cannot be analysed if this cell of interest comes into contact with another cells throughout its recording period / The premise of single cell analysis ensures that fluorescence due to calcium flux is a direct result of material surface interactions and cell-cell modulation is avoided
Upon coming into focus, the fluorescence intensity of Fluo-4 AM (green channel) within the cell must exceed the background fluorescence intensity of the material surface / The background fluorescence can be measured using the Fiji software (ImageJ), and therefore all cells must begin with a ‘resting’ green fluorescence above the material surface to ensure accurate cell reading
Cells are excluded if they come into focus with less than 30 seconds remaining on the total recording period (ie, t = 770 secs) / Based on the analysis of hundreds of responding cells, 30 seconds was the minimal time to first oscillation. For this reason , we allow 30 seconds of adhesion to truly assess if a cell is responding or not responding
If a fluorescence particle (cell or debris) floats into the region of interest of another cell, it cannot be analysed / Fluorescence particles that are moving in and out of the ‘gate’ created for analysing a single cell will interfere with the true fluorescence produced by the cell of interest alone
Responding cells are only accepted if there is a simultaneous and inverse change of fluorescence for both Fluo-4 AM and Fura Red AM channels / In order to account for changes in the focal plane or cell movement, calcium flux is detected as an increase in Fluo-4 AM (green) and simultaneous and equivalent decrease in Fura Red AM (red). Changes in only one fluorescence may not be indicative of cell activation within our system
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Supplementary Table S2. Relative risk valuesa of the rate of calcium release determined through cox regression analysis of individual T cells
anti-CD3+
anti-28(adh) / anti-CD3
+
anti-28(sens) / anti-CD3(Fab) / anti-CD3(Fab)
+
anti-28(adh) / anti-CD3(Fab)
+
anti-28(sens) / anti-28(adh) / anti-28(sens)
1anti-CD3 / 1.1932 / 2.2482 / 4.1740 / 3.6513 / 5.7726 / 4.0347 / 2.1174
1anti-CD3(Fab) / 0.2915 / 0.5281 / 0.8811 / 0.1.3266 / 0.9540 / 0.5187
2anti-CD3 / 1.0594 / 1.4003 / 1.5069 / 1.0594 / 12.5687 / 0.9727 / 2.6115
2anti-CD3(Fab) / 0.7251 / 0.8827 / 1.0858 / 6.3202 / 0.6789 / 1.4956
3anti-CD3 / 1.0446 / 1.4500 / 1.5487 / 1.6948 / 7.8752 / 1.0031 / 2.7264
3anti-CD3(Fab) / 0.6937 / 0.8921 / 1.0448 / 4.1965 / 0.6811 / 1.4464
aRelative risk values were determined through cox regression analysis, and are compared to groups whereby values >1 are increased and values <1 are decrease rate of calcium release.
1Segment 1 represents comparisons from graphs of time to first oscillation for the total population .2Segment 2 represents comparisons from graphs of time to first oscillation for the responding population. 3Segment 3 represents comparisons from graphs of time between first and second oscillation
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