Sarvesh Varma, Joel Voldman*
Supplemental Information水泥磨
PCR Primers
Serum inductions
Cells were seeded in 6-well plates to reach 80% confluence. Serum free media (0.15% serum) was introduced to cell cultures for 24 hours in order to serum-starve cells prior to induction. The serum st
近视回归镜
arved cells were then incubated with regular cell culture media with 3, 10 and 20% (v/v) serum concentrations for 30 minutes, 1 hour and 2 hour durations. The reference controls were cell cultures with no serum inductions. Cells were lysed for RNA extraction immediately after the serum exposure. Induced expression was normalized to basal expression of serum starved cells. The results are shown in SI Figure 1.
Electronic Supplementary Material (ESI)for Lab on a Chip.This journal is ©The Royal Society of Chemistry 2015
SI Figure 1.GAPDH-normalized gene expression of serum-induced cells at 30 min (A), 1 hour (B) and 2 hour (C) exposure duration. N = 2 experiments, error bar: standard error of mean.
ibm as400
Flow Cytometry Gating and Setup
For the detection channels, PE-TexasRed-YG-A (red channel) filter was used with an excitation wavelength of 561 nm and emission detector centered at 610 nm with a 20 nm bandwidth. The reference channel was chosen to be FITC with an excitation wavelength of 488 nm and its emission detector centered at 530 nm with a 30 nm bandwidth. The flow cytometry experimental template was setup using untransfected cells, stable RFP expressing cells (positive control) and stable YPet expressing cells (secondary reference positive control).扣具
Flow cytometry instrument gains and settings were set in order to capture the dynamic range of expression based on these controls and were used consistently among all flow cytometry experiments. The primary gates first were set to select the majority of cell populations from the forward and side scatter information. The subset of all the events analyzed that qualified as cells were set by the intersection gates named P1, P2, and P3 within the forward and side scatter channels as shown in SI Figure 2A . The combined population from the intersection of P1, P2 and P
3 gates was analyzed for expression of red fluorescence against FITC reference channel. The fluorescence cluster of the untransfected (blank) cell population was centered with an approximate zero mean fluorescence in all channels. A horizontal gate was set above to threshold the maximum background fluorescence from the control population to create a sub-population called P5. The distribution fluorescence intensity was analyzed by a histogram of the red channel. An example of P5 within P1, P2 and P3 gates, as well as the RFP intensity distribution histogram for a non-fluorescent cell population is shown in SI Figure 2B, C. The percentage of cells in the P5 gate was termed ‘% activated cells’ for all the experimental conditions. When comparing the fold induction of percent activated population, or in other words, the shift in the fluorescence distribution histogram, the % activated cells in P5 of the induced population was normalized by that in non-induced population. To analyze the change in the fluorescence upon induction, the mean red intensity of the combined P1, P2 and P3 population was compared from before and after induction. The mean RFP fluorescence of this population after induction w as divided by that of the uninduced population and was termed as the normalized fold RFP induction.
A.
SI Figure 2. A. Forward and side scatter gates chosen in flow cytometry to highlight regions of live cells. B. Gating the auto-fluorescence of blank cells to create P5 region. C. RFP intensity distribution plotted for the entire cell population within the side and forward gates (P1, P2, and P3) in the PE-TexasRed (RFP) channel.
Sorted Clonal cells were induced with PMA and the best clone was selected on the criteria of best induction at the population level. SI Figure 3 shows the RFP intensity histograms for the final chosen clone before and after PMA exposure.
SI Figure 3. Population RFP intensity histograms for control and induced conditions for the clonal population
The fraction of induced cells expressing RFP levels above the threshold of maximum fluorescence level of the control population were compared against PMA doses, and are shown in SI Figure 4.
SI Figure 4.A. Induced fraction of activated cells 24h after PMA exposure. N = 3, error bars: standard error of mean. B. Representative histograms of control cells and PMA treated cells (100 ng/ml for 24h) shows population shift.
Inhibitor studies included blocking PMA based induction of PKC via staurosporine (SI Figure 5) and blocking PMA based induction of PKC and MEK using staurosporine and PD98059 together (SI Figure 6).
SI Figure 5. Inhibition of PMA induction using PKC inhibitor staurosporine. N = 3, error bars: standard error of mean.电厂
脱硫塔
防腐 .
SI Figure 6.Inhibition of PMA induction using PKC inhibitor staurosporine and MEK inhibitor together. N = 3, error bars: standard error of mean.
Microfluidic perfusion device and individual channel geometry is depicted in SI Figure 7.
A.
B.
SI Figure 7. A. Image of microfluidic device for FSS studies. B. Channel geometry and dimensions.
豫公网安备41160202000603
站长QQ:729038198
关于我们
投诉建议