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I have been working with a FITC labelled IgG2b MoAb from a commercial
source. It comes lyophilized and I reconstitute it according to
manufacturers recommendations. I then make appropriate dilutions in
PBS/0.1% Azide and store dark in a refrigerator. Storage has been less than
90 days.
I vortex the stock before making dilutions and I vortex the MoAb dilutions
before each use. I do a mononuclear cell prep over percoll and suspend in
PBS/0.1% Azide/ 0.4% FCS working at 4C. I use a microcentrifuge to do 3
washes with the above mentioned suspension media and finally resuspend in
fixative of PBS/0.1% Azide/ 0.4% FCS/ 2% Formaldehyde.
I would like to know from the field, your preferences on :
1. MoAb handling and media for dilutions and storage
is there any problem with storage in plastic?
2. Media and conditions for cell suspension and incubation
is the use of a microcentrifuge for short periods (2min) a problem?
3. MoAb handling when bringing from storage to working conditions
is vortexing a potential problem?
4. Any problems working over Percoll gradients? we changed manufacturers
near failure time and old percoll is temporarily unavailable?
I have spoken to several individual researchers and, of course, the company
but with some conflicting answers and still no experimental effect, I would
appreciate all comments and help. I won't change the color of my eyes until
I hear replies.
Thank you! Khris
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web