Endy:NPE flow cytometer: Difference between revisions

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==Sheath fluid==
==Contact People==
The special marine diluent functions as sheath fluid and resuspension medium when analyzing bacterial cells. It has a high osmolarity (~733 mosM according to M.Brochu Sr. at NPEsystems); compare with ~300 mosM for typical saline like 1xPBS. The high osmolarity permits better signal on the electronic volume channel for size < 1um; lower osmolarity would give noisier results. A concern, however, is that a high osmolarity solution will lead to shrinkage of the cells as compared to normal growth medium. Diluting the diluent can help mitigate this effect, but will probably increase noise. If you plan to study cells with volume > 1um3, the special marine diluent may be diluted by ~30% and still give acceptable (not results. However, I haven't tried that yet. Another solution is to run cells immediately after resuspension in the Special Marine Diluent.
*[[Barry Canton]]
*[[Sriram Kosuri]]
*[[Francois St-Pierre]]


==Calibration Spheres==
==Usage notes==
We currently have spheres which can be used to:
*[[Endy:NPE flow cytometer sheath fluid|Sheath fluid]]
(1) make sure the fluorescent channel(s) are behaving properly
The special marine diluent functions as sheath fluid and resuspension medium when analyzing bacterial cells. It has a high osmolarity (~733 mosM according to M.Brochu Sr. at NPEsystems); compare with ~300 mosM for typical saline like 1xPBS. The high osmolarity permits better signal on the electronic volume channel for size < 11&mu;m<sup>3</sup>; lower osmolarity would give noisier results. A concern, however, is that a high osmolarity solution will lead to shrinkage of the cells as compared to normal growth medium. Diluting the diluent can help mitigate this effect, but will probably increase noise. If you plan to study cells with volume > 1&mu;m<sup>3</sup>, the special marine diluent may be diluted by ~30% and still give acceptable (not results. However, I haven't tried that yet. Another solution is to run cells immediately after resuspension in the Special Marine Diluent.  
(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


The calibration we currently have (August 2005) were purchased from [http://www.spherotech.com Spherotech]. Other labs use spheres from [http://www.polysciences.com Polysciences] or [http://www.bangslabs.com Bangs Labs]. Michael Brochu (Sr, I believe) said he uses 1um spheres with cat.#FS03F from Bangs Labs which give 7-8% CV (on the fluorenscence or the EV channel?]. I was unable to find that particular product on Bangs Labs website, though.
*[[Endy:NPE flow cytometer Calibration spheres|Calibration spheres]]

Revision as of 21:31, 10 August 2005

Contact People

Usage notes

The special marine diluent functions as sheath fluid and resuspension medium when analyzing bacterial cells. It has a high osmolarity (~733 mosM according to M.Brochu Sr. at NPEsystems); compare with ~300 mosM for typical saline like 1xPBS. The high osmolarity permits better signal on the electronic volume channel for size < 11μm3; lower osmolarity would give noisier results. A concern, however, is that a high osmolarity solution will lead to shrinkage of the cells as compared to normal growth medium. Diluting the diluent can help mitigate this effect, but will probably increase noise. If you plan to study cells with volume > 1μm3, the special marine diluent may be diluted by ~30% and still give acceptable (not results. However, I haven't tried that yet. Another solution is to run cells immediately after resuspension in the Special Marine Diluent.

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