Anti-Clara Cell Secretory Protein Antibody

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Recovered cells were trypsinized in 0.05% Trypsin-EDTA (Invitrogen) and resuspended at a concentration of 1×107 cells in 100 µl PBS with 3% FBS.	Two microliters of the rabbit anti-CCSP antibody (Millipore, Billerica, MA) was added, followed by a 30 min incubation on ice. Cells were washed twice in PBS with 3% FBS, then 2 µL goat anti-rabbit- FITC secondary antibody was added and incubated on ice for 30 min. After two washes in PBS with 3% FBS, cells were resuspended in the same but fresh media. Rabbit IgG staining was used as an isotype-matched negative control and CCSP staining with permeabilization of dissociated cells was used as a positive control.

Upstream tips
Recovered cells were trypsinized in 0.05% Trypsin-EDTA (Invitrogen) and resuspended at a concentration of 1×107 cells in 100 µl PBS with 3% FBS.
Protocol tips
Two microliters of the rabbit anti-CCSP antibody (Millipore, Billerica, MA) was added, followed by a 30 min incubation on ice. Cells were washed twice in PBS with 3% FBS, then 2 µL goat anti-rabbit- FITC secondary antibody was added and incubated on ice for 30 min. After two washes in PBS with 3% FBS, cells were resuspended in the same but fresh media. Rabbit IgG staining was used as an isotype-matched negative control and CCSP staining with permeabilization of dissociated cells was used as a positive control.

Publication protocol

Recovered cells were trypsinized in 0.05% Trypsin-EDTA (Invitrogen) and resuspended at a concentration of 1×107 cells in 100 µl PBS with 3% FBS. Two microliters of the rabbit anti-CCSP antibody (Millipore, Billerica, MA) was added, followed by a 30 min incubation on ice. Cells were washed twice in PBS with 3% FBS, then 2 µL goat anti-rabbit- FITC secondary antibody was added and incubated on ice for 30 min. After two washes in PBS with 3% FBS, cells were resuspended in the same but fresh media. Rabbit IgG staining was used as an isotype-matched negative control and CCSP staining with permeabilization of dissociated cells was used as a positive control. CCSP positive (CCSP+) and negative (CCSP−) fractions were obtained by fluorescence-activated cell sorting (FACS) using Vantage SE® cell sorter (BD, Bedford, MA). They were examined by immunfluorescence, adherent or 2D and sphere cell (3D) cultures and qRT-PCR.

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