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I have been sorting with the charge plates upside down !.
(which is incorrect).
My many thanks for the 40 replies that I received from friends and
experts. Here is the combined wisdom in no particular order. Also wish to
thank those who offered their e-mails for more detailed help.
1. Lift up the collection tubes (by means of an O-ring or ball under the
tube) so as to fully engage the stream.
2. The long arms of the charging plates should be UP, towards the nozzle.
There is an error in the older BD manual (1988). Unfortunately, some
people are still sorting with the long arms DOWN (on the standard charging
plates).
3. Sheath Fluid: may not contain enough ions (e.g. if diluted with water).
4. Clean off the place where the nozzle is attached. Salt deposition here
may cause problems. Two thin wires, one for charging the drops and the
others for making the streams vibrate need cleaning.
5. Get the high voltage and circuits checked.
6. The sheath pressure recommended in the BD manual is too high, many
suggested around 10 psi for a 70um nozzle, 27500 Hz, 12 drops, 3V
amplitude.
7. Angle between the charge plates influences the distance between
streams. The angle should be made maximum, about 1/4 inch apart or to
allow a cotton swab to pass through, some suggest a 2-4 mm distance
between the plates. Allow about 30 min atleast for the
plates to "warm" up.
8. The silastic tubing in the fluidics pinch box may sometimes get
blocked. BD part: 80-10005-00, Dow Corning part 602-285, dimensions
0.062 inch ID x .125 inch OD.
9. Wet plates: can cause poor separation. Clean in DI or 70% ethanol,
followed by air drying.
10. Check for bubbles or other obstructions in sample tubing and nozzle.
Samples fixed in ethanol and not washed with buffer can cause problems.
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