Pe conjugated goat anti mouse igg secondary antibody

Cells were dispersed with accutase solution and washed with PBS solution. After blocking nonspecific epitopes with 2% BSA in PBS at room temperature for 30 min, each sample was incubated with 1 μg C38 mouse anti-GRP78 primary antibody for 1 h on ice, with Mouse IgG2b Isotype Control (53,484; Cell Signaling Technology) antibody as the control. Then, the unbound antibodies were rinsed away by three washes with cold PBS, incubating with a PE-conjugated goat anti-mouse IgG secondary antibody (12–4010-82; Invitrogen) at a concentration of 0.25 μg per sample in PBS for 30 min on ice. The samples were washed with PBS, and csGRP78 was detected by C6 flow cytometer and shown as the mean fluorescence intensity in FL2 channel.

Percoll-gradient purified neutrophils from peripheral blood were fixed with 4% paraformaldehyde for 30 minutes at room temperature, and permeabilized with 0.5% Triton X-100/PBS for 30 minutes. The cells were then blocked with Blocking buffer (5% normal goat serum in 0.1% Triton X-100/PBS) for 1 hour, and incubated with the CFTR-specific antibody [42] (link) (mAb #660, CFTR Antibody Distribution Program, UNC/CFF Therapeutics, Inc.) or IgG2b isotype control antibody in Blocking buffer for 2 hours at room temperature. Next, the cells were washed with PBS and subsequently incubated with the PE-conjugated goat–anti-mouse IgG secondary antibody (Invitrogen, Camarillo, CA, USA) for 45 minutes. Expression levels were analyzed by flow cytometry.