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 2n=
d
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 (o=
r 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 be=
low.
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 v=
ery
positive for the short UV (400nm ) adjust voltage of PMT3 so that the hi=
stogram
of PARAB (PMT3 AUX2 =3D 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 ru=
n the
cells which has been loaded with INDO-1. Since these cells will be negat=
ive for
the short UV (unless calcium has been activated ie. bound to calcium will=
dramatically reduce the long and increase slightly the short, ratio =3D =
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 1=
0mV 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=A6g/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 cyt=
ometer
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 calculat=
ions
involved. Any suggestions?
Thanks,
Maris
Maris Handley
Dana-Farber Cancer Institute
Boston, Ma 02115
(617)632-3179
handley@sorter.dfci.harvard.edu