Endy:NPE flow cytometer Calibration spheres

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Why we use calibration spheres

We currently have spheres which can be used to:

  1. make sure the fluorescent channel(s) are behaving properly
  2. get a standard to correct day-to-day fluctuations in the readings of the fluorescent channels
  3. calibrate the electronic volume channel (EV) i.e. establish a correspondence between channel number and volume

Spheres we have in the lab

The calibration we currently have (August 2005) were purchased from Spherotech. Other labs use spheres from Polysciences or Bangs Labs. Michael Brochu Sr said he uses 1um spheres with cat.#FS03F from Bangs Labs which give 7-8% CV (on the fluorenscence or the EV channel?].

How to dilute the spheres

The spheres should be diluted so that the rate is < 500 counts/sec. Higher rates increase the chance of doubles. Note that I checked a couple of blanks (medium without any cells) and they gave counts (noise) at a rate of ~1-2 counts per second. This mean that the lower the count rate of your spheres, the higher the % noise. I try to get the rate between 200 and 500.

Here are dilutions of our current batch of spheres (2005-08-23) which give adequate rates:

  • 0.87um spheres (Spherotech): 10^-4 dilution in Isodiluent (NPE systems) gives a rate of ~200counts/sec
  • 1.79um non-fluorescent polysterene beads (Spherotech): 0.5*10^-4 dilution in Isodiluent (NPE systems) gives a rate of ~ 250 counts/sec.