Extended Data Fig. 4. Alum particles traffic to lymph nodes and are internalized by antigen presenting cells in vivo.
(a) Structure of IR680-pSer4 conjugate, synthesized by Cu-free click chemistry to directly label alum. (b) pSer-dye labeling of alum is stable even following incubation in serum. IR680-pSer4 conjugate was incubated either alone or with alum for 30 minutes, and then 10% mouse serum was added, and the solution was incubated at 37 °C for 72 hours. Data represents the fluorescence measurements of the supernatant after centrifugation to remove any dye remaining bound to alum. Other dyes (Cy3-DBCO and AlexaFluor488-DBCO) were conjugated in the same manner. (n=3 samples/group) Center lines and error bars represent mean and standard deviation, respectively. (c, d) Groups of BALB/c mice (n=5/group) were immunized with 5 µg IR800 dye-labeled Ser4-eOD (c) or pSer8-eOD (d), each mixed with 50 µg IR680-labeled alum, and total fluorescence from dLNs was measured in excised tissues at serial time points. Center lines represent mean. (e–h) BALB/c mice (n=5 mice per group for naïve and day 3 groups, n=4 mice per group for day 7 groups) were immunized with pSer8-eOD:alum or Ser4-eOD:alum by s.c. injection, and flow cytometry was performed on draining lymph nodes after 1 or 7 days. Alum was labeled with pSer4-AF488, and eOD was labeled with AF647. Center lines and error bars represent mean and standard deviation, respectively. (i) ICP-MS measurements of aluminum in inguinal LNs of naïve mice and mice that were immunized s.c. 3 or 8 days prior to measurement. (n=5 mice/group). Center lines and error bars represent mean and standard deviation, respectively.