#Flowjo table editor

[passionpiner]

Flowjo table editor

Select a sample that you want the number of molecules for. Copy the derived parameter to the All Samples group.(An a/b symbol appears beneath your sample.) Derive Parameters window, showing the parameter definition. x is the parameter being used to measure the number of molecules, andįigure 8.The derived parameter should equal the definition of a line, y = mx + b, where: Enter the slope of the line from Step 19.Ĭlick the “+” button, and add the intercept from Step 19.Click the Multiply button, or add an asterisk to the nascent expression.Select the parameter used for the calibration (for example, FITC).Just below the plot, in the formula panel, click Insert Reference.In the Derive Parameters menu, enter a name for the parameter (for example, the No.From the drop-down menu, select Derive Parameters.In the workspace, right-click on a sample.Correlation Plot, showing slope and intercept. (You can save the image, or leave the plot open.)įigure 7. Note the slope of the line and the intercept.(If they’re reversed, simply click Transpose Axes.) Ensure the target fluorochrome is on the X-axis and the “No.In the Plots band, click the Correlation Plot button. In the Table Editor, highlight both entries.Table Editor, showing the original and new entry. The Table Editor should now have two entries, the MFI statistic and the “No.Add Column dialog, showing the File Keywords pane. Select the keyword you added in Step 2 from the list of keywords in the left pane, and click OK.įigure 5.Add Column dialog, showing the Keyword tab. In the Add Column dialog window, click the Keyword tab.įigure 4.From the Columns band, select Add Column. Drag in the MFI statistic node into the Table Editor.Add the median or geometric mean statistic (MFI) to one of the gated populations, and copy it to the group.įigure 3.Move the ranged gates in the remaining samples to their appropriate positions.Copy the gate to the group (Command + Control + Shift + G).Graph window, showing a ranged gate on the histogram’s modal population. Create a ranged gate on the modal (peak) population.įigure 2.Change the plot to a histogram with the primary channel on the X-axis.Open the sample representing the calibration blank.(These should be known values provided by the manufacturer, for example 8,000, 16,000, 64,000, and so on.) In the workspace, add the appropriate values to the “No.Sample window, showing new keyword column. (Note: If you have a keyword/value pair that corresponds to the number of molecules on the cell, you can skip this step and the next)įigure 1. (Note: if your calibration standards were acquired as one tube, first export the individual peaks, and then re-import the new FCS files into FlowJo). Place your calibration standard samples into their own group.The following steps guide you through creating the standard curve, calculating the line that fits the curve, and ultimately deriving the number of molecules on the surface of a cell in your experiment: Have three or more standards that cover the anticipated range of expression on your target cells, together with a blank. In FlowJo v10, we need to start with data from your calibration standards. The strict measurement being determined here is the molecules of equivalent fluorescence (MESF). Note: In the following example, we assume one bound antibody per molecule, which may not be true depending on antibody class, distance between molecules, and number of targeted epitopes on a given molecule. With the standard curve we derive a linear relationship between fluorescence intensity and number of molecules on a given cell. Considering that fluorescence intensity is correlated with molecules on the surface of the cell, can the relationship between the two be quantified? If so, how can we use that relationship to calculate the number of molecules on the surface of a cell in a given experiment?Īs with all indirect measurements, a standard curve must first be created using calibration standards (for example, cytometric bead arrays), to establish the relationship between the fluorescence intensity measurements and the antibody binding to its target molecule. Measuring the fluorescence intensity of cells and particles is routine and the basis of the vast majority of inquiry in flow cytometry.

#Flowjo table editor

[equitytonki]

Flowjo table editor

Flowjo table editor how to#

Flowjo table editor password#

Flowjo table editor download#

FYI, if you followed everything exactly up to here, you should only have 2 data columns and not 3. To actually use this data elsewhere (such as with R), export it into a csv format which can be easily imported by other programs. This will make a new window, holding the table you wanted. With the settings fixed, you can hit the “Create Table” button at the top of the main workspace. To do this, double click on that row, and select the statistic you want to include.Īnd you should now have something that looks like this: For example, I find it much more informative to have the singlets show total count, rather than Freq of parent. Some of those statistics aren’t what we’re looking for. Once in this window, select the populations / statistics you want to include from the main workspace, and drag it into the table editor, so you have something that looks like this. Lastly, the easiest way to output this data is to hit the Table Editor button near the top of the screen to open up a new window.

Flowjo table editor how to#

Of course, a scatterplot needs a second axis, so I just used mCherry fluorescence (or the lack of it, since these were just normal 293T cells), captured by the YL2-A detector.Īnd of course copy that to the group as well (you should know how to do this by now). While this can be done in histogram format, I generally also do this with a scatterplot, since it allows me to see even small numbers of events (which would be smashed against the bottom of the plot if it were a smoothed histogram). In this case, it’s GFP positivity, captured by the BL1-A detector. Next is actually setting up the analysis for the response variable we were looking to measure. Then only gate the cells directly on the diagonal, thus excluding those that have more FSC-A relative to FSC-H. To do this, make a scatterplot where FSC-A is on the X-axis, and FSC-H is on the Y-axis. Here is the best description of doublet gating I’ve seen to date. So the live cell population will already be enriched for singlets, but having a second “doublet gating” step will make it that much more pure. Once you do that, the population should now be in bold, with the same text color as the group name. Thus, right click on the “Live” population in the workspace and hit “Copy to Group”. Once you have made that gate, you’ll want to keep it constant between samples. Since we’re mostly using HEK cells, that means that main thing we will be doing in this screen is gating for the population of cells while excluding debris (small FSC-A but high SSC-A). Set it so forward scatter (FSC-A) is on the X-axis, and side scatter (SSC-A) is on the Y-axis. By default, a scatterplot should show up. the first sample), and double clicking on it. Start by choosing a representative sample (eg. Now to actually start analyzing the flow data. Now that the group is made, I select it, and then drag the new sample files into it, like below: Thus, I hit the “Create Group” button and type in the name of the group I’ll be analyzing. Before you start dragging in samples, I find it useful to make a group for the specific set of samples you may want to analyze. With that out of the way, now you can perform your analysis. Thus, click on the “Preferences” button:Īnd finally once in that final window, change the “Decimal Precision” value to something like 8. This can be prohibitively uninformative if you have very low percentages that you’re trying to accurate quantitate. Once logged in, you’ll be starting with a blank analysis workspace, as below.īefore I forget, an annoying default setting of FlowJo is that it only lists two decimal points in most of its values.

Flowjo table editor password#

I’m obviously not going to share my login and password here ask someone in the lab or consult the lab google docs.

Flowjo table editor download#

Obviously, if you don’t have FlowJo yet, then download it from the website. Thus, here’s a short set of instructions for using it to do a basic process, such as determining what percentage of live cells are also GFP positive. While I’ve been using FlowJo for a long time, I realize it isn’t super intuitive and new people to the lab may first struggle in using it. Inevitably, what ends up being the most practical tool for analysis of low cytometry data is FlowJo. We do a lot of flow cytometry in the lab.

#Flowjo table editor

[justdepot]

Flowjo table editor

First, there is a limit to how brightly-stained the original population can be seven divisions results in cells that have 1/128th (your MFI, Count, or Frequency statistics very easily and quickly. The Table Editor is often used to export a table of statistics into Excel, Numbers, and even Prism. Click OK and your column definition will be added to the Table Editor.If you have chosen the %ile, specify the value in the Percentile box.Select the parameter of the population from which to calculate the statistic.If the statistic is to be applied to a subpopulation, select it from the Population drop-down menu.Choose a sample from the drop-down menu that typifies the target population.Scroll through the list and select the statistic you wish to apply.To create a table column that applies a statistic to the samples in the current group using the interface above, take the following steps: This method is much more complicated and we encourage you to simply drag the statistics from the workspace. When the Column Information dialog appears, click the Statistic tab. In the Columns band, click “Add Columns”. When the table is created, it will batch automatically.Īlternatively, you can click the Edit tab in the Table Editor. *Please note: when you batch your statistics to the group as you see in the example workspace above, you only need to drag prototypic statistics from the FIRST sample. The easiest way to populate a table with statistics is to drag them from the workspace window to the Table Editor. In each case you can name the column, define it as a control value, choose whether or not to display its values in the finished table, and format the output according to criteria of your choice. There are three types of columns you can create when you click the “Add Column” button in the Table Editor: Statistic (described below), Keyword, and Formula columns. The main function of the Table Editor - exporting statistics!

#Flowjo table editor

[internationallong]

Flowjo table editor

#Flowjo table editor how to#

First, there is a limit to how brightly-stained the original population can be seven divisions results in cells that have 1/128th (of these values have biological relevance. Neither assay could be used to determine the precursor frequency (i.e., what fraction of original cells ever underwent division) nor the distribution of the number of divisions that responding cells underwent. Cell counting provides an assessment of the total expansion of the culture (i.e., how many cells are present at termination compared to initiation) 3H-T provides an assessment of how many cells were in cell cycle during the pulse phase (usually the last few hours prior to termination). Prior to the introduction of this assay, there were two dominant methods for quantifying proliferation: cell counting and 3H-thymidine ( 3H-T) uptake. The basic principle is well-described, and relies on the two-fold dilution of fluorescence accompanying each cellular division as the fluorescently-tagged cellular proteins are allocated equally to each daughter cell. A flow cytometric assay based on the dilution of carboxy fluorescein diacetate succinimidyl ester (CFSE, also known as CFDA-SE) fluorescence during culture has become the standard for this purpose ( 1) when done well, analysts can enumerate cells that have divided as well as quantifying how many divisions each cell has undergone ( 2- 4). Stay tuned.An important assessment of cellular function, particularly in immunology, is the proliferative capacity of cells under different conditions. In the next post, I'll talk about the formula editing in the table tool, which pretty much incorporates basic spreadsheet operations directly into FlowJo. Notice the four line items near the bottom - these are the settings that can be tweaked to make your table reporting more powerful. But how can these settings be customized? To find out, drag a gate or stat node from the workspace window into the table editor, and double click on it: Take a value, examine it, if it meets certain criteria, red flag it.īy defualt FlowJo is set to mark values outside 1 SD from the mean in bold, and 2SD in red. If there was a staining screwup in one well on a 384 well plate, I bet you a beer it's faster to identify it using the table than it is the workspace/graph widnow. Repeatitive analysis tends to accumulate information which can be used to establish certain reproducible trends about the data, such as % expressions of certain markers, or CV's / means of known populations. Graph window(s)

#Flowjo table editor how to#

Table editor manual entry for Mac - clickīasic tutorial demonstrating how to make stats out of the table editor - clickįlowJo's approach to data analysis invites for fast iteration and This post is meant to shed some light on the usability of table editor in that regard. FlowJo for Macintosh Table Editor classier settings are underused.

#Flowjo table editor