Immunofluorescence in undifferentiated PromoCell cardiomyocytes (Line E) shows only diffuse cytoplasmic expression of sarcomeric actinin (green) with little or no co-localization with the actin cytoskeleton (red).
Immunofluorescence in PromoCell cardiomycytes (Line E) differentiated for 4 weeks shows increased focal expression of sarcomeric actinin (green) that strongly co-localizes with adhesions sites and the actin cytoskeleton (red).
| Assay Date(s): | 08/11/2015 |
| Primary Antibody: | Mouse monoclonal anti-alpha-actinin (sarcomeric) (Sigma, Cat: A7811-100UL, Lot: 103M4770) |
| Secondary Antibody: | Alexa Fluor 488 goat anti-mouse (Invitrogen, Cat: A11001, Lot: 1397999) |
| Temperature: | 37ºC |
| Dilution | 1:100 |
| Diluent: | 10% goat serum, 2.5% bovine serum albumin in PBS |
| Actin Staining: | Alexa Fluor 647 phalloidin (Invitrogen, Cat: A22287, Lot: 648322) ) |
| Dilution: | 1:40 |
| Diluent: | PBS |
| Nuclear Staining: | Hoechst 33342, trihydrochloride, trihydtrate (Invitrogen, Cat: H3570, Lot: 46C3-1) |
| Dilution: | 1:10,000 |
| Diluent: | PBS |
| Imaging Date: | 08/11/2015 |
| Imaging Platform: | Zeiss LSM 780, Axio Imager 2 |
| Objective: | Plan-Apo 40x/1.4 Oil DIC M27 |
| Excitation: | 458 (Argon), 633 (HeNe 633), 405 (Diode 405-30) |
| Emission: | 410-508, 490-632, 638-755 |
| Related Standard Operating Procedures (SOPs) | |
| Cell SOPs: | DToxS SOP CE – 1.0: PromoCell Cardiomyocyte Subculture |
| DToxS SOP CE – 2.0: PromoCell Cardiomyocyte Plating for Drug Test | |
| DToxS SOP CE – 4.0: Drug Treatment and Cell Lysis | |
| Assay SOPs: | DToxS SOP A – 4.0 Immunofluorescence PromoCell Cardiomyocytes |
| Performed by: | B. Hu, G. Jayaraman, R. C. Calizo |