Cd4 fitc clone rm4 5

Mice were sensitized to chicken OVA (Sigma-Aldrich, St. Louis, MO, USA) as described [6 (link)]. The mice were then challenged with aerosolized OVA for 30 min once (single challenge) or once a day for 3 days (multiple challenge). Control groups were not sensitized or challenged. Additional groups of mice received i.p. 1, 5, or 10 mg/kg olaparib (Selleckchem, Pittsburgh, PA, USA) in saline 30 min after OVA challenge. AHR, organ recovery, histopathology, bronchoalveolar lavage (BAL), cytokine and OVA-specific IgE assessment, and FACS analysis were performed as described [6 (link), 22 (link), 23 (link)]. To determine CD4⁺ T cell populations, spleens were processed to generate single cell suspensions after which splenocytes were stained with antibodies to mouse CD3e (145-2c11-APC) and CD4-FITC (clone RM4-5) (both from e-Bioscience, San Diego, CA, USA). To determine T-regulatory (T-reg) cell populations, splenocytes were stained with CD4 (GK1.5-FITC) and CD25-APC (clone PC61) (from Biolegend, San Diego, CA, USA), and intracellularly with anti-mouse Foxp3 (FJK-16s)-PE (e-Bioscience) followed by FACS analysis. The multiplex assay and FACS were conducted at the LSUHSC Comprehensive Alcohol Research Center Core.

Spleen tissue was gently mashed through a 70 µm nylon membrane filter in lymphocyte separation medium to obtain single cell suspensions. Lymphocytes were collected following centrifugation. Following cell count and dilution, lymphocytes were treated with anti-mouse CD16/CD32 antibodies (Mouse BD Fc Block, BD Biosciences) for 15 min and then stained with CD4-FITC (clone RM4-5, eBioscience), CD8-PerCP-Cy5.5 (clone 53-6.7, eBioscience), CD62L-PE (clone MEL-14, BD Biosciences), and CD44-APC (clone IM7, eBioscience) antibodies. The intracellular staining of TLR8 (clone 112H7.15, Dendritics) or BCL2 (clone BCL/10C4, Biolegend) was performed according to the instructions of the manufacturer (Fixation/Permeabilization Solution Kit, BD Biosciences). Following staining, cells were washed with PBS, fixed in 1% formaldehyde, and analyzed on BD Accuri C6 Plus or FACSAria (BD bioscience). Frequency of target cell types was calculated by FlowJo software.

Cells collected from brain, spleen, or BM were filtered through 50-μm cell strainer (Partec) and blocked with antibodies against Fc-receptors (CD 16/32, clone 2.4G2; in house). Cells were stained with following anti-mouse antibodies for the time course experiment: CD3e-PeCy7 (clone 145-2c11; eBioscience), TCRβ-APC (clone H57-597; eBioscience), CD4-FITC (clone RM4-5; eBioscience), CD8-BV570™ (clone 53-6.7; Biolegend), CD49b (clone DX5; in house), and Propidium Iodine (Thermo Fisher Scientific) to test cell viability. The myeloid cell population was stained with the following antibodies: CD45.2-APC, CD11b-Pacific Blue (clone M1/70; in house), Ly6G-FITC (clone 1A8-Ly6g; eBioscience), Ly6C-Biotin (clone AL-21, BD Biosciences), CD115-PE (clone AF598, eBioscience), streptavidin-PerCP (BD Biosciences), and Pacific Orange-N-Hydroxysuccinimide (NHS) (in house) as viability dye. Stained live cells were acquired (stopping gate of 100,000 live cells) using a MACS Quant Analyzer 10 and analyzed by FlowJo software.