Anti cd1d 1b1

Single cell suspensions from spleen, liver and visceral adipose tissue were incubated with 2.4G2 mAb (anti-FcγRIII/II) to block non-specific binding of primary mAb and then cells were stained with a combination of the following mAb: FITC conjugated anti-TCRβ (H57-597, BioLegend), anti-CD11b (Mac-1, TONBO Biosciences), anti-CD206 (C068C2, BioLegend), APC conjugated anti-NK1.1 (PK136, BioLegend), anti-CD11c (HL3, BD Pharmingen), PE conjugated anti-α-GalCer:CD1d complex antibody (L363, BioLegend), α-GalCer (PBS-57)-loaded CD1d tetramer kindly provided by NIH Tetramer Core Facility at Emory University (Atlanta) and Brilliant Violet 421 conjugated anti-F4/80 (BM8.1, TONBO Biosciences). 3T3-L1 adipocytes were stripped off by PBS containing 1.5 mM EDTA and stained with PE conjugated anti-CD1d (1B1, BioLegend), anti-CD80 (16-10A1, BD Pharmingen) and -CD86 (GL1, BD Pharmingen). Stained cells were sorted using FACS Aria (BD Biosciences) or assessed using FACS Caliber flow cytometers (BD Biosciences). Data were analyzed with FlowJo software (FlowJo, LLC).

Mice were given i.p. injection of 200 μg of anti-CD1d (1B1, Biolegend) blocking antibody diluted in 200 μL of PBS, and intrapleural injections of 150 μg of anti-CD1d blocking antibody diluted in 60 μL of PBS to each side at 1 dpi. Mice that received injections of isotype-matched antibody served as controls. Mice were sacrificed and lung tissues were collected at 4 dpi. Mice were i.p. injected 200 μg of anti-TCRγδ (GL3, Biolegend) or isotype control antibodies 3 days before pdmH1N1 infection. B-1a cell depletion was achieved using protocols previously reported with some modifications^{71 (link),72 (link)}. Briefly, 6-week-old mice were treated with i.p. injections of 3 mL water daily for two days, and control mice received the same volume of PBS. The next day of the second water injection, mice were given i.p. injection of 200 μg of purified anti-mouse IgM (RMM-1, Biolegend) antibody diluted in 200 μL of PBS, and intrapleural injections of 150 μg of purified anti-mouse IgM (RMM-1) diluted in 60 μL of PBS to each side. Mice that received injections of isotype-matched antibody served as controls. Mice were allowed for 4 days to rest for influenza infection and another 4 days for flow cytometric analysis. The efficiency and specificity of B-1a depletion in various organs were examined at 4 dpi.