Apc anti mouse cd11c clone hl3

NY-ESO-1 was labeled with Alexa Fluor® 594 with the Microscale Protein Labeling Kit (Invitrogen, Carlsbad, CA, USA). The labeled NY-ESO-1 was incubated with DP7 (40 µg/ml), CpG (20 µg/ml), or the CpG/DP7 combination for 10 min and then incubated with a culture of BMDCs for 1 h. Cell membranes tagged with green fluorescent dye. BMDCs were washed three times with PBS, fixed in 4% paraformaldehyde, and stained with DAPI. Finally, the BMDCs were observed under a confocal laser scanning microscope.
To study the effect of the CpG/DP7 complex on BMDCs, BMDCs were incubated with DP7 (40 µg/ml), CpG (20 µg/ml) or the CpG/DP7 combination for 16 h, stained with APC-anti-Mouse CD11c (Clone HL3; BD Biosciences Cat# 550261), FITC-anti-mouse-CD40 (Clone 3/23; BD Biosciences Cat# 561845), PerCP-Cy5.5-anti-mouse-CD80(clone 16-10A1; BD Biosciences Cat# 560526) or FITC-anti-mouse-CD86 (clone GL1; BD Biosciences Cat# 561962). BMDCs were gated as CD11c⁺ and analyzed for CD40, CD80, CD86 expression by NovoExpress software (ACEA Biosciences, Inc.).

Cultured BMMs were detached as described by Han et al. [14 (link)]. The BMMs were resuspended at 1.0 × 10⁶ cells/1 ml in PBS and then incubated with FITC-anti-mouse CD86 (clone GL1, TONBO Biosciences, San Diego, CA), PE-anti-mouse CD80 (clone 16-10A1, TONBO), Alexa Fluor^® 647-anti-mouse CD206 (clone MR5D3, BD Pharmingen). For immunoprofiling of tumor-bearing mice, single cell suspensions of Hepa1-6 tumors or the draining lymph nodes (dLN) at 1 × 10⁶ cells/sample were immunostained with a combination of some of the following antibodies, such as FITC-anti-mouse CD4 (clone GK1.5, Tonbo), PE-anti-mouse CD11b (clone M1/70, BD), PerCP-Cy5-anti-mouse-B220 (clone RA3-6B2, Tonbo), APC-anti-mouse-CD11c (clone HL3, BD), eFluor450-anti-mouse CD45 (clone 30-F11, eBioscience), APC-Cy7-anti-mouse-CD8 (clone 53-6.7, Tonbo), eFluor450-anti-mouse CD44 (clone IM7, eBioscience), APC-anti-mouse CD62L (clone MEL-14, eBioscience), eFluor450-anti-mouse CD8 (clone 53-6.7, eBioscience), and eFluor660-anti-mouse CD107a (clone 1D4B, eBioscience). Data of the stained samples were acquired using an LSR II flow cytometer (BD) and analyzed using the software FlowJo (Tree Star Inc., Ashland, OR).

Antibodies against STAT3, phosphorylated STAT3 (p-STAT3), C-Rel, and p-C-Rel were purchased from Bioss (Woburn, MA). WST-1 cell proliferation assay kit was obtained from Roche Diagnostics (Mannheim, Germany). FITC Anti-mouse B7-1 (Clone 16-10A1), PE Anti-mouse B7-2 (Clone GL1), PE Anti-Mouse IL-12 p40/p70 (Clone C15.6) and APC Anti-mouse CD11c (Clone HL3) antibodies were purchased from BD Pharmingen (San Diego, CA). IL-6 and IL-12 ELISA kit were obtained from R&D System Inc. (Minneapolis, MN). Anti-IL-6 (ab6672) and anti-CD11c (ab33484) antibodies for immunofluorescent (IF) staining were purchased from abcam (San Francisco, CA). Anti-IL-12 (NB600-1443) antibody for IF staining was purchased from Novusbio (Littleton, CO). Mouse sIL-6R alpha Simple Step ELISA Kit (ab203360) was obtained from abcam (Cambridge, MA). Cell Fixation/Permeabilization Kits (BD554715) for intracellular cytokine analysis was purchased from BD Bioscience (San Jose, CA). Purified Rat Anti-mouse IL-6 antibody (clone MP5-20F3) was obtained from BD Biosciences. STAT3 inhibitor VI, S31-201 was purchased from Santa Cruz Biotechnology.