Product Manual (SG05)
Protocol for Combined Analysis (SG05 and C544)
This product is a simple detection kit by plate assay for senescence-associated β-galactosidase (SA-β-gal) activity which is used as a marker for senescent cells. By simply adding SPiDER-βGal, a reagent for detection of β-galactosidase, to 96 well plates, this kit allows you to quantify SA-β-gal activity and makes it possible to evaluate multiple specimens.
When normalization is done by the results obtained by counting cells, quantifying nucleic acids (the relevant product), or quantifying proteins, the measured values obtained using this kit become available for evaluating SA-β-gal activity according to cell number.
Simply Quantify Senescent Cells:
Cells prepared in advance are lysed in the buffer supplied with this kit.Fluorescence intensity is obtainable according to SA-β-gal activity simply by adding the fluorescent substrate SPiDER-βGal to the cell lysate.
Even when you prepare cells in 100 mm dishes or others, fluorescence intensity can be measured by transferring cell lysate in 96 well plates after cell lysis.
Correlation with imaging data:
Imaging assessments of WI-38 cells at different passage levels were performed with this Plate Assay Kit and the Cellular Senescence Detection Kit - SPiDER-βGal
As a result, it was confirmed that in both kits, SA-β-gal staining increased in the high-passage WI-38 cells.
Bear in mind that although initial cell seeding densities are the same, cell densities at the time of plate assay differ due to low proliferation rate of senescent cells at higher passage levels. Therefore, in this experiment, we used SA-β-Gal activity values normalized by the results obtained using the Cell Count Normalization Kit (coming soon) in which cell number is determined by a nuclear marker.
Ex. 535nm / Em. 580nm
Green：Ex. 488nm / Em. 500-600nm (SA-β-Gal staining with Cellular Senescence Detection Kit - SPiDER-βGal(Item# SG03))
Blue：Ex. 405nm / Em. 450-495nm (Nuclear staining with -Cellstain- DAPI solution(Item# D523))
Evaluation doxorubicin-treated with cells:
We performed plate assays using this kit with WI-38 cells after adding doxorubicin which increases the production of mitochondrial reactive oxygen species (ROS). As a result, it was confirmed that the addition of doxorubicin to WI-38 cells caused increased staining intensity of SA-β-gal due to DNA damage-induced senescence.
<Condition> Ex. 535nm / Em. 580nm
Precautions when using this kit:
Cell counts may need to be normalized. When cells are analyzed in a microplate, the results obtained may sometimes differ depending on cell numbers per well.In such cases, normalization of the measured values obtained from cell counting and total protein will be necessary. In this kit, cell numbers can be easily measured by the fluorescence intensity induced by a reagent added to cell culture medium for staining nuclei.
(Normalization kit is now available) >>
Product page to Normalization Kit (Cell Count Normalization Kit)