Parameter is the channel in which the antibody signal was collected 9. Population is the gate from which to calculate the statistic (e.g. Sample can be any of the tubes from the current group (antibody) being analyzed 7. Choose Median from the list of available statistics 6. In the Edit ribbon, click Add Column to open the Column Information window 4. Double-click on the name of the table and change it to indicate the antibody in question 3. Separation Index = Start by adding the necessary statistics to the table: MedianPositive MedianNegative (84%Negative MedianNegative)/ Click Table Editor in the FlowJo ribbon to open the FlowJo Tables window 2. The higher the Separation Index value, the better the separation between positive and negative populations. Each term in the equation refers to a statistic derived from the fluorescence intensity of the antibody staining. FlowJo for Antibody Titrations: SI and Concatenation UWCCC Flow Lab 05/05/16 KF 1.0ΔΆ Calculating Separation Index in a Table The Separation Index (SI), defined by the equation below, is a metric for evaluating the results of the staining. Changes made in one group will apply to that sample in all groups. No-Antibody Control Note that the sample with no antibody (viability dye only) will need to be gated for each group in turn. It may be necessary to adjust the Positive and Negative gates for each sample if there are large shifts in the populations. Then draw broad gates for the Positive and Negative populations. Gate Populations Within each group, gate for live, single cells. If samples are organized by manually dragging them into groups, the listed order comes from the order in which samples are added to the group. It is also possible to add a custom Keyword (not described here) to determine the order. Then click in the new Time column to order samples based on that value. Right-click on the column names and choose Edit Columns to access a window in which the Time column can be added. A straightforward way to order samples is by time. ![]() Sample Order The order of samples within a group will determine the order of those samples in the final concatenated dot plot derived later in these instructions. Each group may also contain the viability only or no antibody or unstained sample. ![]() Groups can be defined with Sample Inclusion Criteria to automatically grab samples, or simply named and populated manually by dragging files from All Samples into the newly created groups. 1 FlowJo for Antibody Titrations: Separation Index and Concatenation UWCCC Flow Cytometry Laboratory Highland Ave 7016 WIMR Madison, WI Group Samples by Antibody After importing the FCS files into FlowJo, create a group for each antibody in the experiment.
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