Pe conjugated anti mouse cd80

Alexa Fluor® 700 anti-mouse CD11c (clone N418), anti-Mouse MHC Class I (H-2Kb) eFluor® 450 (Clone: AF6-88.5.5.3), FITC conjugated anti-mouse/rat MHC Class II (I-A/I-E; clone M5/114.15.2), APC anti-mouse CD40 (clone 1C10), PE conjugated anti-mouse CD80 (clone 16-10A1) antibodies with their respective isotype controls comprising of Alexa Fluor® 700 conjugated Armenian hamster IgG (clone eBio299Arm), FITC rat IgG2b κ (clone eB149/10H5), Rat IgG2a k Isotype Control APC (clone eBR2a), PE-conjugated Rat IgG2a (clone eBR2a), Rat IgG2a k Isotype Control PE-Cyanine7 (clone eBR2a), and mouse IgG2a k Isotype Control eFluor® 450 (clone eBM2a) were purchased from eBioscience^™ (San Diego, CA). PE/Cy7^™ rat anti-mouse CD86 (Clone GL1) was purchased from BD Pharmingen^™. PE/Cy 5.5 anti-mouse CD205 (MMR, clone NLDC-145) and PerCP/ Cy5.5 Rat IgG2a, κ Isotype control were procured from BioLegend®.

After treatment for 24 hours, BMDCs were harvested and stained with fluorescein FITC-conjugated anti-mouse CD11c, PE-conjugated anti-mouse CD11b, PE-conjugated anti-mouse CD80, PECy5-conjugated anti-mouse CD86, and PECy5-conjugated anti-mouse major histocompatibility (MHC) class II antibodies (eBioscience). The FITC Annexin V apoptosis detection kit (BD Pharmingen) was used for cell death studies. Cells were washed with PBS and fixed with 1% paraformaldehyde (Merck). Fluorescently labeled cells were analyzed by flow cytometry (FACScan, BD).

Lymph nodes and minced tumors were digested using a cocktail of 1.67 Wünsch U/mL Liberase TL (Roche) and 0.2 mg/mL DNAse (Roche) as previously described [40 (link),64 (link)]. After lysis of red blood cells single cell suspensions were filtered through a 40-μm nylon cell strainer. Live cells were distinguished using Fixable Viability Dye eFluor 660 (eBioscience) prior to flow staining. All samples were incubated with anti-mouse CD16/32 (Fc block) for 10 minutes followed by staining for various surface markers for 30 minutes at 4°C. The following antibodies were used (all purchased from eBiosciences): PE-Cy7-conjugated anti-mouse CD11c, Alexa Fluor 700-conjugated anti-mouse CD86, PE-conjugated anti-mouse CD80, PE-conjugated anti-mouse CD1d, PE-conjugated anti-mouse CD70, FITC-conjugated anti-mouse CD40 and Alexa Fluor 700-conjugated anti-mouse MHC Class I (H-2Kd/Dd), FITC-conjugated anti-mouse CD27, PECy7-conjugated anti-mouse CD3ε, APC-conjugated anti-mouse CD122, Alexa Fluor 700-conjugated anti-mouse CD4. iNKT cells were identified by staining with mouse CD1d/PBS-57 or CD1d/unloaded tetramers (NIH Tetramer Core Facility) for 30 minutes at room temperature [65 (link)]. All samples were analyzed using a BD LSR II flow cytometer and FlowJo version 8.7.5 (Tree Star).