Biomedical Engineering Reference
In-Depth Information
3. Use median rather than mean data.
4. Export FCS fi les as FCS 3.0:
(a) Gated events, e.g., P2 FITC.
(b) File version, e.g., FCS 3.0, not FCS 2.0.
(c) Parameter type: tick “none” for all parameters and “linear”
for only the width readouts required, e.g., FITC-W.
(d) Save to the experiment folder (will create another folder
within that folder).
5. Use FCS extract 1.2 to convert exported FCS 3.0 data into a
format that can be recognized by Excel:
(a) Select “batch extraction.”
(b) Select directory where data folder was stored.
(c) Select parameters required, e.g., you may have exported
DAPI, FITC, and 568, but only need to process the
FITC data.
(d) Select process batch—data will disappear as it is processed.
(e) Excel (comma-separated data) will be saved into the same
folder.
6. Open data in Excel.
1. Create an Excel spreadsheet template of bin ranges (e.g., 0,
500, in 500 increments to 300,000). Use a formula to add
+500 down a column.
2. Copy in columns of data from the process batch, e.g., 0 min,
30 min of a time point, triplicates of conditions, and untreated
vs. treated data.
3. Calculate the frequency of bins using the Histogram analysis
tool in Excel:
(a) Click on “Data” tab at the top of Excel page.
(b) Click on “Data Analysis” tab.
(c) Choose “Histogram” from the list.
(d) Select one of your data columns as the “Input Range”—
note do not choose empty cells.
(e) Select your bin column as your “Bin Range”—note do not
choose empty cells.
(f) Select an empty cell at the top of an empty column for the
“Output Range”—this is where the generated data will
be placed.
(g) Click “OK.”
4. The bin and frequency will be generated in the empty column.
5. Label columns so that you do not lose track of which data is
being processed.
3.4.2 Generation
of Frequency Table
