[Cytometry] CD5 in a lymphoproliferative panel

Jáksó Pál jakso.pal at pte.hu
Thu Apr 25 03:05:13 EDT 2013


In my experience the higher background is inherent with these dyes and comes from data spreading caused by spectral overlap. As CD5 is  usually dim in B-CLL it's better to use more "sensitive" dyes. These dyes may give nice separations if they are not or minimally affected by spectral crosstalk between the dyes in the experiment, but their background may look awful if there is a significant overlap from other channels. It would be useful if you could give more details about the staining combination you use.

However you have some choices:

1. you may try CD5 FITC, or PE instead. We use CD5 FITC (clone MEM-32 from Exbio).

2. try to use such staining combinations in the spectrally overlapping channels which are not supposed to be expressed on the CD5 positive cells.
For example in my experience it's better to omit PerCP-Cy5.5 staining if I want to measure dim APC signal.

3. An other possibility: DNA stains in the red channels can increase the background if they are not removed from the system correctly from previous measurements. Especially live cell stains, e.g. DRAQ5 which can stick to the sample port, flow cell and tubing wall and stains the cells in seconds. We always do 0.5% bleach washing every times after DRAQ5.
This problem can be checked easily if you measure unstained cells and look on the affected channels intensities vs. time. If you see increasing signal in the first 30 second and then reach a plateau you may suspect that some live permeant DNA dye stains your cells.
We had hard times some years ago when DRAQ5 was introduced and we didn't know why we have high background in the red channels. Detergent based cleaning solutions what we used for regular cleaning were not enough to remove this dye.

These are just some possibilities...

Best regards,

Pal Jakso, PhD
biologist
Flow Cytometry Laboratory
Dept. of Pathology
Faculty of Medicine
University of Pecs
12. Szigeti str.
H-7624 Pecs
HUNGARY

On Apr 24, 2013, at 6:04 PM, "Dalal, Bakul [Dr.][VA]" <Bakul.Dalal at vch.ca>
 wrote:

> We have recently experienced high bkg in CD5 using clone L17512. We use CD5 APC, PE-Cy7 and PerCP-Cy5.5, and all three give the same high bkg (APC least bkg > PerCP-Cy5.5 > PE-Cy7). Can anyone share his/her experience?
>
> Do you have a clone that works well?
>
> Many thanks.
>
> Bakul I. Dalal MD FRCPC FCAP FASCP
> Clinical Professor, Faculty of Medicine, Univ British Columbia
> Director, Flow Cytometry Service
> Division of Hematopathology, Vancouver General Hospital
> Vancouver, BC, Canada
> bakul.dalal at vch.ca<mailto:bakul.dalal at vch.ca> / BakulDalal at gmail.com<mailto:BakulDalal at gmail.com> / +1 (604) 875-4496 (O) /+1 604 779 6968 (C)
>
>
>
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