fitc conjugated anti cd40  (Bio-Rad)

Journal: Journal of Immunology Research

Article Title: Conventional DCs from Male and Female Lupus-Prone B6.NZM Sle1/Sle2/Sle3 Mice Express an IFN Signature and Have a Higher Immunometabolism That Are Enhanced by Estrogen

doi: 10.1155/2018/1601079

Figure Lengend Snippet: Estrogen enhances the upregulation of cDC activation markers in response to TLR stimulation. Bone marrow precursors from B6 (black closed symbols) or TCSle (gray open symbols) female (circle) and male (triangle) mice were cultured with GM-CSF in standard phenol red/media conditions or media depleted in phenol red and void of steroids (charcoal-treated FBS: 0 E2) supplemented with 0.03 nM, 0.1 nM, or 50 nM E2. On day 7, cDCs were stimulated with CpG (10 μ g/mL) (b, e, h) or R848 (1 μ g/mL) (c, f, i). cDCs were harvested 24 hours post stimulation, stained, and gated on singlets, live cells, and CD11c + CD11b + (gating shown in ) before analyzing costimulatory molecules (a–i). Representative histogram plots of CD40, CD86, and CD80 on unstimulated (black line) or CpG-stimulated (gray histogram) female TCSle cDCs from standard conditions (j). Mean + SE values are from 3 (female cDCs) or 4 (male cDCs) independent experiments, using one mouse of each strain and sex per experiment. Two-way ANOVA analysis with Tukey multiple comparisons was used to calculate the significance of the effects of E2 treatment within each group of mice, represented by brackets below the graph. Black ∗ indicates significance within all 4 groups while symbols represent significance within a single group. Two-way ANOVA analysis with Tukey multiple comparisons was used to compare differences between B6 and TCSle, and results are shown in a box surrounding the symbol Δ for significance between B6 and TCSle males or the symbol O for significance between B6 and TCSle females. ∗ , Δ, and O represent p < 0.05. ∗∗ , ΔΔ, and OO represent p < 0.01. ∗∗∗ represents p < 0.001 and ∗∗∗∗ p < 0.0001.

Article Snippet: cDCs were harvested 24 h post stimulation, washed in cold PBS, incubated with rat anti-mouse CD16/CD32 (clone 2.4G2, BioLegend) mAb for 10 min to block Fc γ Rs, and then stained for 30 min on ice with allophycocyanin-conjugated hamster anti-mouse CD11c (N418, eBioscience), PE-Cyanin7-conjugated rat anti-mouse CD11b (M1/70, BD Bioscience), either PE-conjugated rat anti-mouse CD86 (GL1, BD Bioscience) or PE-conjugated rat anti-mouse CD115 (AFS98, BioLegend), FITC-conjugated hamster anti-mouse CD80 (16-10A1, eBioscience) or FITC-conjugated hamster anti-mouse CD40 (HM40–3, BD Biosciences), and FITC-conjugated rat anti-mouse MHCII (M5/114.15.2 eBioscience) or Biotin-conjugated rat anti-mouse MHCII (M5/114.15.2 eBioscience) followed by streptavidin PerCPCy5.5 (eBioscience) or Biotin-conjugated goat anti-mouse MERTK (polyclonal R&D Systems) followed by streptavidin FITC.

Techniques: Activation Assay, Cell Culture, Staining