The Premier Platform for Single Cell Analysis (1) Titrations in Cytobank To analyze data from a titration in Cytobank, the first step is to upload your own FCS files or clone an experiment you already have access to. To practice using one of our approved datasets, log on to cytobank.org, find the CD4 titration experiment, and use the Clone FCS files link in the experiment overview to obtain an unannotated copy of the experment. After cloning the experiment, go to the Working Illustration and begin gating the data by clicking on Gate under the Populations figure dimension. (2) Our goal is to use internal positive and negative cell subsets as controls. So we will use CD4+ lymphocytes (CD45+ CD3+) as a positive control, use CD4 dim monocytes (CD45+ CD14+) to check for sensitivity for a dim population, and use B cells (CD45+ CD20+) as an internal negative control that does not express CD4. (1) First, set the axes to Cell_length vs Ir193 and draw a gate similar to the picture shown. Use the Check Gates button to ensure that the gate is appropriate for all samples. Change the Active Population to the gate Intact cells. (2) Next, change the y-axis to CD45, draw a gate around the CD45+ events, and change the Active Population to CD45+. *While we use medians in this guide, there may be other reporting methods that are more appropriate for your data such as 5-to- 95 percent distance or Stain Index. 1
(3) (4) (3) Set the axes to CD3 vs CD20 and gate the B cells (CD3- CD20+) and CD3+ Lymphs (CD3+ CD20-). (4) Make the CD3+ Lymphs the Active Population and change the axes to CD4 vs CD8a. From the File/Sample Name pulldown menu, select the CD4_0_cells_found file and draw a CD4 single positive gate containing 1% of the negative events. 2
(5) (6) (5) While CD3+ Lymphs is still the Active Population, change the axes to CD8a vs CD20 and draw a gate around the CD8a- cells and call it CD3+ CD8a- (this is for showing the titration in a figure later). (6) Set the axes to CD45 vs CD14 and change the Active Population to CD45+. Draw a gate around the CD14+ events and name it Monocytes. 3
(7) Apply and Return to the Working Illustration, click on Conditions to turn on the figure dimension, and click on Setup to tag files with our staining conditions. In the Add Conditions box, type in 0, 0.03, 0.1, 0.3, 1.0, 3.0 and click Add Conditions. Cytobank will automatically sort the files into the correct bins. After you confirm that the files were sorted correctly, return to the Working Illustration. (8) From here, click Choose under Populations and select only the CD3+ CD8- population. Click Choose under Channels and select only CD4. On the left side, change the y-axis to CD45 and then select the Illustration tab and Click here to update the Illustration. You should now see results similar to the figure below: Use the text box in the upper left corner of the Working Illustration to save this figure as CD3+ Dot Plots. After the dot plots have been saved, return to the Working Illustration. (9) Move the Conditions figure dimension to the middle position and use the Plot Type pulldown menu to change the plot from Density Dot to Histogram Overlay. Click here to update. (10) Save this new figure as CD3+ Histogram Overlay. 4
(11) After the figure has been saved, you ll be brought to the Experiment Overview page. From this location, click on Export Statistics Tool and Define What to Export. (12) First, add the Conditions annotation: Click Add Statistic -> Medians -> CD4+ Lymphs -> CD4 -> Add column, and do the same for Monocytes, and B cells, adding the median of the CD4 channel for each. If you desire, you can also add the Percent of CD4+ Lymphs that are staining positive by clicking Add Statistic -> Percent in Gate -> CD3+ Lymphs -> CD4+ Lymphs -> Add column. (12) (13) Once all the desired statistics are added to the table, click Export. Once the calculations are finished, download the resulting file (visible in the Experiment Overview under Exported Data Files) and open in a spreadsheet program (Google Docs, Excel, OpenOffice, etc). (14) The goal is to determine the difference between the signal (your positive population) and the noise (the non-specific binding of the antibody). To do this, divide the CD4 Medians of the CD4+ Lymphs by the CD4 Medians of the B cells on a line chart with the Conditions as the x-axis and the medians as the y-axis. Use this chart or the original medians to determine what the optimal concentration of antibody is for your sample by selecting the concentration of antibody with the largest distance between the negative and positive populations. 5
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