Calibrate arbitrary channels to molecules-of-fluorophore using fluorescent beads (eg, the Spherotech RCP-30-5A rainbow beads.)
Computes a log-linear calibration function that maps arbitrary fluorescence units to physical units (ie molecules equivalent fluorophore, or MEF).
To use, set Beads to the beads you calibrated with (check the lot!) and Beads File to an FCS file containing events collected using the same cytometer settings as the data you’re calibrating. Then, click Add a channel to add the channels to calibrate, and set both the channel name and the units you want calibrate to. Click Estimate, and make sure you check the diagnostic plot to see that the correct peaks were found.
If it didn’t find all the peaks (or found too many), try tweaking Peak Quantile, Peak Threshold and Peak Cutoff. If you can’t make the peak finding work by tweaking , please submit a bug report!
The beads you’re calibrating with. Make sure to check the lot number!
- Beads file
A file containing the FCS events from the beads.
A list of the channels you want calibrated and the units you want them calibrated in.
- Peak Quantile
Peaks must be at least this quantile high to be considered. Default = 80.
- Peak Threshold
Don’t search for peaks below this brightness. Default = 100.
It works best to choose units of a fluorophore that is spectrally
matched to the channel that you’re calibrating. Here are the beads
Cytoflow knows about (including the laser and filter sets
used to characterize the beads):
Spherotech ACP 30-2K
Spherotech RCP-30-5A Lot AN04, AN03, AN02, AN01, AM02, AM01, AL01, AK04, AK03 & AK02**
Spherotech RCP-30-5A (Euroflow) Lot EAM02 & EAM01
Sphreotech RCP-30-5A (Euroflow) Lot EAK01, EAG01, EAE01 & EAF01
Spherotech RCP-30-5A Lot AK01, AJ01, AH02, AH01, AF02, AF01, AD04 & AE01
Spherotech RCP-30-5A Lot AG01
Spherotech RCP-30-5A Lot AA01, AA02, AA03, AA04, AB01, AB02, AC01 & GAA01-R
Spherotech RCP-30-5A Lot AC02, AC03 & AD01
Spherotech RCP-30-5A Lot Z02 and Z03
Spherotech RCP-30-5 Lot AA01, AB01, AB02, AC01 & AD01
Spherotech RCP-30-5 Lot AM02, AM01, AL01, AH01, AG01, AF01 & AD03
Spherotech URCP 38-2K
Spherotech URCP 38-2K Lot AN01, AM01, AL02, AL01, AK03, AK02, AK01, AJ02 & AJ03
Spherotech URCP 50-2K Lot AM01 & AJ01
Spherotech URCP 50-2K
The Spherotech fluorophores labels and the laser / filter sets (that I know about) are:
MECSB (Cascade Blue, 405 –> 450/50)
MEBFP (BFP, 405 –> 530/40)
MEFL (Fluroscein, 488 –> 530/40)
MEPE (Phycoerythrin, 488 –> 575/25)
MEPTR (PE-Texas Red, 488 –> 613/20)
MECY (Cy5, 488 –> 680/30)
MEPCY7 (PE-Cy7, 488 –> 750 LP)
MEAP (APC, 633 –> 665/20)
MEAPCY7 (APC-Cy7, 635 –> 750 LP)