If mice are given one single dose (0.5mg) of anti-Ly6G (1a8) IP to deplete neutrophils, how long will it take for the neutrophils to be replenished to near normal levels?
Dear Evan,
We tested this using 0.1 mg 1A8 (BioXCell) i. p. (Day 0). We saw an approx. 80% depletion which lasted until Day 5. Neutrophil numbers started to increase on Day 6 and returned to normal on Day 7. Higher doses of 1A8 did not cause much more severe depletion in our hands but may last a few days longer (we did not test it). You should be careful about how to stain for neutrophils in the presence of depleting doses of 1A8 since the large circulating 1A8 concentration blocks the binding of additional (e. g. fluorescently labeled) 1A8 molecules to the epitope. We used an Ly6B (7/4) antibody to label neutrophils and gated out monocytes based on FSc/SSc. The 7/4 mAb works fine even if neutrophils are saturated with 1A8. You may also consider using the NIMP-R14 mAb for neutrophil depletion. It gave us much more robust (practically complete) depletion which lasted for 3-5 days. The specificity also seemed to be OK. You can look up Weber et al., J Exp Med 2015 (in press; link below) for further details.
Best wishes,
Attila
http://jem.rupress.org/content/early/2014/12/09/jem.20130062.abstract
Dear Evan,
We tested this using 0.1 mg 1A8 (BioXCell) i. p. (Day 0). We saw an approx. 80% depletion which lasted until Day 5. Neutrophil numbers started to increase on Day 6 and returned to normal on Day 7. Higher doses of 1A8 did not cause much more severe depletion in our hands but may last a few days longer (we did not test it). You should be careful about how to stain for neutrophils in the presence of depleting doses of 1A8 since the large circulating 1A8 concentration blocks the binding of additional (e. g. fluorescently labeled) 1A8 molecules to the epitope. We used an Ly6B (7/4) antibody to label neutrophils and gated out monocytes based on FSc/SSc. The 7/4 mAb works fine even if neutrophils are saturated with 1A8. You may also consider using the NIMP-R14 mAb for neutrophil depletion. It gave us much more robust (practically complete) depletion which lasted for 3-5 days. The specificity also seemed to be OK. You can look up Weber et al., J Exp Med 2015 (in press; link below) for further details.
Best wishes,
Attila
http://jem.rupress.org/content/early/2014/12/09/jem.20130062.abstract
Thank you Attila. I have read about neutrophils being coated with anti-LY6G antibodies which necessitates alternative markers like the ones you mention. Do you have any ideas why these opsonized neutrophils are not eliminated?
No idea, Evan. In our hands, it's only about 20% of the normal neutrophil count. It may be that they are in some phase of being recognized and on their way to disappear. The 1A8 mAb is also of a different IgG isotype which may make elimination less efficient. For us, this whole issue was more of a methodological question, not how the elimination occurs and why some cells can apparently escape it. Based on the literature, we were surprised that we did see so many neutrophils still present in 1A8-treated mice (not to mention that 1A8 from a different vendor was much less efficient at even higher mAb doses). The Daley et al. (J Leukoc Biol 2008) paper shows more robust depletion (though they used 0.5 mg/mice, as I understand). However, it is not clear how they identified neutrophils in 1A8-treated animals. To avoid that uncertainty, we decided to use the 7/4 mAb for staining.
I attempted two experiments with the 1A8 antibody from BioXcell and did blood films (with Diff-Quick stain) to visually inspect for neutrophil depleteion (three doses of 0.5mg per mouse over a week) and it did not work very well in our hands. Too expensive to try again to optimize......
That is odd.. I ordered it from BioXcell as well and it is quite potent. I began with a single 200ug i.p. and that was too much (depleted PMNs for over a week, even the PMN in the marrow were opsonized). 10ug depletes for a day or so before the numbers rebound rapidly. I had ordered 25mg of the antibody and it will likely last years at this rate.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Immunology Techniques