Flow Cytometry Antibodies Verified by WB, IF and IHC

Since flow cytometers measure fluorescence as total intensity of a cell, information about the signal's (i.e., antibody) distribution on the cell is lost. Fluorescence microscopy can be used to examine the distribution of the fluorescence signal on, or in a cell and often assists in distinguishing background from truly positive cells. Western blot can be used to obtain confidence about the antibody's specificity. Immunohischemical (IHC) staining can be used to confirm the proper tissue distribution, which also provides information on specificity of the antibody.

For more information about IF/ICC, WB and IHC please view our corresponding webpages.

For example, the human CD147 MAb was verified by WB, IF and IHC:

Antigen: Human CD147
Antibody: CD147 / EMMPRIN / Basigin (10186-H08H)
Cell: A431
WB: 30 ug/lane
Recommended Concentration: 5-10 µg/ml
Secondary Antibody: Dylight 800-labelled Antibody to Rabbit IgG (H+L), at 1:5000 Dilution
Antigen: Human CD147
Antibody: CD147 / EMMPRIN / Basigin (10186-H08H)
Cell: HeLa
IF: 4% PFA in PBS, rt 15min, rabbit anti-CD147 monoclonal antibody (15 µg/ml) at 4℃ overnight, counterstained with DAPI (blue)
Recommended Concentration: 5 µg/ml Secondary Antibody: Alexa Fluor® 488-conjugated Goat Anti-rabbit IgG (green), 1:500
Instrument: Nikon 80i
Antigen: Human CD147
Antibody: CD147 / EMMPRIN / Basigin (10186-H08H)
Cell: Human gastric cancer epithelium cell
IHC: 0.2 µg/mL, formalin-fixed paraffin embedded sections, antigen retrieval with citrate buffer (pH 6.0), micro-oven 15 min
Recommended Concentration: 0.1-5 μg/ml
Instrument: Nikong 80i,200×

Flow Cytometry / FACS Background

Flow cytometry is a method to evaluate cell membrane proteins and intracellular proteins as well as peptides and DNA. The principle behind FACS is an antigen-antibody reaction, with the antibodies being fluorescently labelled. There are three fluorescent proteins (R-PE, APC, and PerCP) conjugated to antibodies. Flow cytometry quantification is carried out with intercalating color labels (without the antibody). Flow cytometry antibodies are widely used in cell counting, cell sorting, biomarker detection and protein engineering.