Goat anti mouse igg1 pe

Freshly isolated NK cells were stored overnight at 4°C in porcine NK medium with 25% fetal calf serum (FCS) and cultured NK cells were collected from the 96-well plates after 60 h of incubation before analysis. NK cells were washed in PBS-EDTA with 1% FCS (further referred to as staining medium) and incubated for 30 minutes with 0.1% Fixable Live/Dead stain (catalog n. L34963, Invitrogen, Carlsbad, CA, USA). Cells were transferred to 96-well conical bottomed plates and incubated for 30 minutes at 4°C with primary antibodies or isotype as listed in Table 2. After incubation, cells were washed, stained with goat anti-mouse IgG1-PE (catalog n. P21129, Invitrogen, Carlsbad, CA, USA) for 30 minutes at 4°C. Cells were washed again and resuspended in 100 µl staining medium for analysis. Flow cytometry was performed using a Beckman Coulter Cytoflex and samples were analyzed using CytExpert software (Beckman Coulter, Brea, CA, USA).

N-term FimH surface expression was detected by flow cytometry. GAD31, GAD40, GAD80, and GAD83 were grown to exponential phase from overnight cultures and washed with PBS. rLA was resuspended in PBS and colony forming units (CFU) calculated based on the optical density at 600 nm. Then, 1 × 10⁷ CFU rLA were suspended in staining buffer (PBS + 1% fetal bovine serum (FBS)) plus mouse anti-FimH mAb824 (a gift from Dr. Svgeni V. Sorurenko, University of Washington, Seattle, WA) followed by goat anti-mouse IgG1 PE (P-21129; Invitrogen, Waltham, MA, USA). Flow cytometry was performed using a Gallios flow cytometer (Beckman Coulter, Indianapolis, IN, USA). Analysis was performed using FlowJo software (Ashland, OR, USA). Gates were set using rLA incubated with only the secondary antibody.