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. Author manuscript; available in PMC: 2016 Jun 1.

Published in final edited form as: J Neuroimmune Pharmacol. 2015 May 16;10(2):318–332. doi: 10.1007/s11481-015-9611-3

O-1966 increases the percentage of Tregs in the MLR. To determine if O-1966 induces Tregs in the MLR, cultures were analyzed by flow cytometry for live (LIVE/DEAD® dead cell stain negative) CD4⁺CD25⁺Foxp3⁺ Tregs. Panel A shows representative scatterplots of MLR cultures harvested at 48 hours and stained with isotype controls and left untreated or pretreated with 32 μM O-1966 or ethanol vehicle, stained with antibodies for CD25 and Foxp3. The cells were gated on live CD4⁺ cells and are expressed as a percentage of total live CD4⁺ cells. Panel B shows the average number of CD25⁺Foxp3⁺ Tregs as a percentage of total live CD4⁺ cells from 3 experiments from cultures left untreated ( ) or pretreated with 32 μM O-1966 ( ) or ethanol vehicle ( ), and harvested at time zero, or after 24 or 48 hours in culture. Data are the mean ± S.E.M. of 3 separate experiments. *p < 0.01. (ANOVA, O-1966 versus vehicle).

Inline graphic — O-1966 increases the percentage of Tregs in the MLR. To determine if O-1966 induces Tregs in the MLR, cultures were analyzed by flow cytometry for live (LIVE/DEAD® dead cell stain negative) CD4⁺CD25⁺Foxp3⁺ Tregs. Panel A shows representative scatterplots of MLR cultures harvested at 48 hours and stained with isotype controls and left untreated or pretreated with 32 μM O-1966 or ethanol vehicle, stained with antibodies for CD25 and Foxp3. The cells were gated on live CD4⁺ cells and are expressed as a percentage of total live CD4⁺ cells. Panel B shows the average number of CD25⁺Foxp3⁺ Tregs as a percentage of total live CD4⁺ cells from 3 experiments from cultures left untreated ( ) or pretreated with 32 μM O-1966 ( ) or ethanol vehicle ( ), and harvested at time zero, or after 24 or 48 hours in culture. Data are the mean ± S.E.M. of 3 separate experiments. *p < 0.01. (ANOVA, O-1966 versus vehicle).