Flow cytometry Anti-bodies Mouse - CCSP

Flow cytometry is an immunophenotyping technique whereby sing cell suspensions are stained for either cell surface markers or intracellular proteins by fluorescently-labelled antibodies and analyzed with a flow cytometer, where fluorescently-labelled molecules are excited by the laser to emit light at varying wavelengths, which is then detected by the instrument. There are several key criteria which are required to be kept in mind while designing a flow experiment- 1. Antibody titration (optimal dilution of antibodies should be calculated in order to avoid over- or under- saturated signals for proper detection of surface and intracellular markers), 2. Precision (3 or more replicates of the sample should be used per experiment), 3. Specificity (proper isotype controls should be included in the experiment), 4. Day-to-day variability (experiments should be repeated 3 or more times to ensure consistency and avoid variability due to flow cytometer settings), 5. Antibody interaction (Fluorescence minus one or FMO should be used, which is the comparison of signals from panel minus one antibody vs. the full panel), and 6. Antibody stability (fluorescently-labelled antibodies should be stored at 4C).

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Found 2 matching solutions for this experiment

Anti-CCSP - Rabbit

Seven Hills Bioreagents

Upstream tips	Protocol tips	Downstream tips
Primary lung cells were isolated from 1- to 12-week-old wild-type SPC H2B-GFP or β-actin-DsRed mice, as previously described (10)	Staining with 7-aminoacrinomycin D (Molecular Probes, Eugene, OR) or 4′,6-diamidino-2-phenylindole (Sigma Chemical Co., St. Louis, MO) staining to eliminate dead cells. Cells were fixed with BD Cytofix/Cytoperm (PharMingen), according to the manufacturer’s instructions. Rat IgG2b and rabbit IgG (PharMingen) were used for isotype controls.	Cell sorting was performed with a Cytomation MoFlo (Beckman Coulter, Inc., Brea, CA) or a BD FACS Aria (BD Bioscience, San Jose, CA), and data were analyzed with FlowJo software (Tree Star, Inc., Ashland, OR).

Upstream tips
Primary lung cells were isolated from 1- to 12-week-old wild-type SPC H2B-GFP or β-actin-DsRed mice, as previously described (10)
Protocol tips
Staining with 7-aminoacrinomycin D (Molecular Probes, Eugene, OR) or 4′,6-diamidino-2-phenylindole (Sigma Chemical Co., St. Louis, MO) staining to eliminate dead cells. Cells were fixed with BD Cytofix/Cytoperm (PharMingen), according to the manufacturer’s instructions. Rat IgG2b and rabbit IgG (PharMingen) were used for isotype controls.
Downstream tips
Cell sorting was performed with a Cytomation MoFlo (Beckman Coulter, Inc., Brea, CA) or a BD FACS Aria (BD Bioscience, San Jose, CA), and data were analyzed with FlowJo software (Tree Star, Inc., Ashland, OR).

Publication protocol 1 Relevant paper

Anti-Clara Cell Secretory Protein Antibody

Sigma-Aldrich

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.

Manufacturer protocol Publication protocol 1 Relevant paper

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