Immunofluorescence Analysis of GR Expression in MDA-MB-231 Cells

MDA-MB-231 breast cancer cells were seeded in 1 × 10⁵ cells per mL and incubated for 24 h. PsA-D or dexamethasone treatment comprised 30 min. Cells were fixed afterwards with −10 °C cold methanol. Cells were made permeable using 0.1% Triton™ X-100. Antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA): primary antibody (sc-8992 GR (H-300)) incubated 1:50 for 24 h overnight at 4 °C and secondary antibody (sc-2012 IgG-FITC (fluorescein isothiocyanate)) was incubated 1:100 for 2.5 h at room temperature. Cells were washed three times with PBS following each incubation step. For staining, cell nuclei 4′,6-Diamidin-2-phenylindol (DAPI, Sigma) was incubated for 5 min at room temperature at a concentration of 3 µM and washed three times with PBS for 5 min.
Quantification of immunofluorescence intensity was achieved with ImageJ (v1.51k). The shape of the cells was outlined and the area, mean gray fluorescence value and integrated density measured. Several background readings were also measured. The “total corrected cellular fluorescence” (=TCCF) was calculated according to following formula: integrated density—(area of selected cell x mean fluorescence of background readings) [63 (link)]. Values of GFP staining were subtracted by values of DAPI staining to obtain cytoplasmic TCCF.