Fitc conjugated anti cd3

An anti-TRPA1 antibody (1:500 dilution) was obtained from Novus Biologicals (Littleton, CO, USA). The selective TRPA1 antagonists HC-030031 (HC) and TCS-5861528 (TCS) were purchased from R&D Systems (Minneapolis, MN, USA). Wheat germ agglutinin (WGA, 1:200 dilution) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for T-CaMKII (1:1000 dilution), calcineurin (1:1000 dilution), GAPDH (1:2500 dilution), α-smooth muscle actin (α-SMA, 1:500 dilution), CD3 (1:200 dilution), and CD68 (1:200 dilution) were purchased from Abcam (Cambridge, MA, USA). An antibody for p-CaMKII (1:1000 dilution) was purchased from GeneTex (Irvine, CA, USA). Antibodies for CD206 (1:200 dilution) were purchased from R&D Systems (Minneapolis, MN, USA). Fluorescein isothiocyanate (FITC)-conjugated anti-CD206, allophycocyanin (APC)-conjugated anti-F4/80, phycoerythrin (PE)-conjugated anti-CD45 and FITC-conjugated anti-CD3 were purchased from BioLegend (San Diego, CA, USA). Secondary antibodies and goat anti-rabbit IgG were obtained from LI-COR Biosciences (Lincoln, NE, USA). All other chemicals were of analytical grade.

Peripheral blood was obtained from the orbital sinus of anesthetized mice. Mononuclear cells were isolated from peripheral blood using erythrocyte lysate. FITC-conjugated anti-CD3 (Biolegend) and PerCP-Cy5.5-conjugated CD19 (BD Biosciences) antibodies were used to label B cells. For labeling, 10⁵ cells were incubated with antibodies at 4°C for 1 h, washed twice, and centrifuged (1000 rpm, 10 min, 4°C). Labeled cells were analyzed on a FACSCanto II flow cytometer using Cell QuestPro acquisition software. In addition, data were analyzed using FlowJo 7.6.1 software.

FACS analysis was performed as described previously.^{30 (link)} Briefly, single-cell suspensions of four LGs from each condition were prepared by treating minced tissue fragments with 100 U/ml collagenase D and 15 μg/ml DNase (Sigma-Aldrich) for 40 min at 37 °C. After blocking with 1 μg unlabeled anti-FcrR antibody (clone 2.4G2), cells (1 × 10⁶) were washed with RPMI-1640 and surface-stained with PE-Cy5-conjugated anti-CD11b, PE-conjugated anti-PI (eBioscience, San Diego, CA, USA), FITC-conjugated anti-CD3, PE-conjugated anti-7-AAD (Biolegend, San Diego, CA, USA), or FITC-conjugated anti-Annexin V (eBioscience), and then analyzed by FACS (FACSCalibur, Becton-Dickinson, Mountain View, CA, USA). For the separation of CD45⁺ and CD45⁻ cells in LGs, FACS ARIA (BD) was used after APC-conjugated anti-CD45 staining (eBioscience).

The following antibodies to human proteins were used: from BioLegend, fluorescein isothiocyanate (FITC)-conjugated anti-CD3 (OKT3), anti-CD14 (HCD14), anti-CD16 (3G8), anti-CD19 (HIB19), anti-CD34 (581), anti-CD94 (DX22), anti-CD123 (6H6), anti-FcER1α (Fc epsilon receptor I alpha) (AER-37); phycoerythrin (PE)-conjugated anti-ICOS (CD278, C398.4A), anti-NKp44 (P44-8), anti-VEGFR2 (7D4-6); brilliant violet (BV) 421-conjugated anti-CD161 (HP-3G10), BV421 anti-CD45RO (UCHL1); allophycocyanin (APC)-conjugated anti-CCR6 (29-2L17), anti-CCR7 (4B12), anti-CXCR5 (J252D4), anti-LFA-1 (M17/4), anti-NRP1 (12C2), and APC-cy7 anti-CD45RA (HI100); from BD Biosciences, PE-CF594-conjugated anti-CD3 (UCHT1) and anti-CD62L (DREG-56); from Becton Dickinson: FITC-conjugated anti-CD34 (581), anti-TCRαβ (IP26), and TCRγδ (B1); from Beckman Coulter, phycoerythrin-Cy7-conjugated anti-CD127 (R34.34) and PE Cy5.5-conjugated anti-CD117 (104D2D1); and from Miltenyi, PE-conjugated anti-NRP1 (AD5-17F6). For phenotypic analyses by flow cytometry, data were collected with an LSRFortessa instrument (BD Biosciences) and analyzed with FlowJo software (Tree Star).