Actually, on all of our exps. we setup our ELITE-ESP using air cooled HeCad
laser with the following procedure. Once the equipment is verified the next
step is to run a biological control ( we stimulated cells with CD2/2R, usually
a delayed response) and a membrane channel control (positive control, we use
ionomycin). Also note that INDO loading will be different with different cells
so you may need to adjust your PMT voltage to accommodate this fact.
Calcium INDO-1 exp. Procedural set-up
In the DNA check program:
+ Using the DNA check beads align lasers / not x axis positioning of the laser
cofocal lens sharpens the peak - timing from 488 laser was 30msec.after
adjustment of the y and z knob of the HeCad lens.
+ Using the DNA check beads run both lasers and check PMT2 or PMT3 for 2nd
signal from HeCad laser. (HV must be increased to see 2nd peak).
+ Check timing and gates for all signals when the gated amp is disabled. After
enable the gated amp and check signals. All signals should be within the GATE!
In the Calcium program (INDO/Calcium 042997) :
+ Use the standard Blue beads (R&D systems - 324/421nm) dilute 1/1000 (or until
the rate of 300 can be established) and run using the calcium program and
filters as discribed by the protocol.
+ Adjust forward scattter and ninety degrees as described in the table below.
Normal PBMC+s Blue Beads
Forward Scatter 400 / 3 930 / 15 *
L Ninety Degrees 316/10 336/10
*This change represents the fact that these beads are very small.
+ Run blue beads - these beads will be negative for long UV (500nm) but very
positive for the short UV (400nm ) adjust voltage of PMT3 so that the histogram
of PARAB (PMT3 AUX2 = long UV) on the x axis and PMT1 (short UV) is 60 degrees
from the x axis or from the long UV. Or the PMT1 voltage should be 10mV as
viewed on the ocilloscope.
Final Voltage Final Gains
PMT1 (Short UV) 700 10
PMT3 (PARA-AUX2)
Long-UV) 560 10*
*AUX?PARMB gains was 4.
+ Return the FS and L90 degrees back to the normal PMBC+s settings and run the
cells which has been loaded with INDO-1. Since these cells will be negative for
the short UV (unless calcium has been activated ie. bound to calcium will
dramatically reduce the long and increase slightly the short, ratio = short /
long. Hence when bound to calcium the ratio will increase mostly because of the
decrease in the long UV) but positive for the long UV adgust voltage to 10mV as
viewed on the ocilloscope. This should be approx. a 10 degree look when viewing
the PMT1 vs. PARMB (x axis).
+ Experimental cells can now be tested - the procedure should include a clear
negative and positive stimulas such as CD2/2R or Ionomycin (10¦g/ml).
Stephen P. Perfetto
HIV Diagnostic Laboratory
Walter Reed Army Institute of Research
1600 East Gude Drive
Rockville, MD. 20850
_______________________________________________________________________________
Subject: INDO-1 Calibration
From: HANDLEY@sorter.dfci.harvard.edu at Internet_Gateway
Date: 6/16/97 10:27 AM
Hello Everyone!
Does anyone know if there is a kit available to help calibrate a flow cytometer
for calcium flux using Indo-1? I have a researcher who wants to be able to
compare values between experiements, and I have suggested using ionophore
and EGTA to help with this, but he seems reluctant to do all the calculations
involved. Any suggestions?
Thanks,
Maris
Maris Handley
Dana-Farber Cancer Institute
Boston, Ma 02115
(617)632-3179
handley@sorter.dfci.harvard.edu
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