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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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