Journal: Diabetes

Article Title: Endosomal Sequestration of TLR4 Antibody Induces Myeloid-Derived Suppressor Cells and Reverses Acute Type 1 Diabetes.

doi: 10.2337/db21-0426

Figure Lengend Snippet: Figure 8—TLR4-Ab remains colocalized with EEA1 and sequesters TLR4/MD2 inside the endosome for at least 24 h. BMDMs were pulsed with LPS (100 ng) or TLR4-Ab (UT18 100 ng) for 5 min, washed, and then fixed at the noted time points. Cells were stained (CD11b, TLR4/ UT18, EEA1) for imaging flow cytometry, and 5,000 events (gated on CD11b1 cells) were collected for each cell population. A and B: Rep- resentative images for LPS- and TLR4-Ab–stimulated BMDMs are shown for each time point. C: Internalization analysis generated using IDEAS software provides a mean internalization erode score using CD11b as the surface marker and TLR4/UT18 as the internalization probe. TLR4 internalization scores >1.0 indicate internalization of TLR4. P values calculated in GraphPad Prism. *P < 0.05, ****P < 0.0001. D: Bright detail similarity (median) measures pixel-by-pixel correlation between TLR4 and EEA1 in each image, effectively measur- ing colocalization of the two probes at each denoted time point. Bright detail similarity >1.0 indicates colocalization of TLR4 and EEA1. *P 5 0.014, **P 5 0.0067. The quantification of C and D represent data collected from three independent experiments.

Article Snippet: BMDMs were plated in 24-well plates at 2 × 106 cells/mL in complete DMEM with Fc block (1:5,000; BioLegend), then pulsed with TLR4-Ab (UT18 100 ng), Ctrl-Ab (UT15 100 ng), or LPS (100 ng) for 5 min. BMDMs were stained in FIX & PERM (Invitrogen) with antibodies against CD11b (BioLegend), EEA1 (Novus Biologicals), and UT18/UT15-specific secondary antibodies (BioLegend).

Techniques: Staining, Imaging, Cytometry, Generated, Software, Marker