Abstract
Cell quantification is widely used in basic or applied research. The current sensitive methods of cell quantification are exclusively based on the analysis of non-fixed cells and do not allow the simultaneous detection of various cellular components. A fast, sensitive and cheap method of the quantification of fixed adherent cells is described here. It is based on the incubation of DAPI- or Hoechst 33342-stained cells in a solution containing SDS. The presence of SDS results in the quick de-staining of DNA and simultaneously, in an up-to-1,000-fold increase of the fluorescence intensity of the used dyes. This increase can be attributed to the micelle formation of SDS. The method is sufficiently sensitive to reveal around 50–70 human diploid cells. It is compatible with immunocytochemical detections, the detection of DNA replication and cell cycle analysis by image cytometry. The procedure was successfully tested for the analysis of cytotoxicity. The method is suitable for the quantification of cells exhibiting low metabolic activity including senescent cells. The developed procedure provides high linearity and the signal is high for at least 20 days at room temperature. Only around 90 to 120 minutes is required for the procedure’s completion.
Highlights
Cell quantification is a common task for many laboratories
As the reduction of MTT leads to the creation of insoluble formazan crystals which have to be solubilised with e.g. DMSO, other tetrazolium-based assays such as 3-(4,5-dim ethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and 4-(3-(4-iodop henyl)-2-(4-nitrophenyl)2H-5-tetrazolio)-1,3-benzene disulphonate (WST-1) were developed[1,4]
As there is no need for cell lysis, the cell cycle analysis by image cytometry or the detection of various cellular components can be performed before the elution step
Summary
Cell quantification is a common task for many laboratories. A typical example of its use is drug-discovery research. Cell lysis is usually required for maximal sensitivity and the linearity of the dependence of the signal on the cell number. It can result in the substantial prolongation of the procedure and/or additional costs. We have developed an approach for the quantification of the fixed cells that does not require cell lysis as the DNA dye is eluted from cellular DNA using an elution solution It results in a highly homogeneous solution of the eluted DNA dye and substantial increase of its fluorescence intensity. It provides the possibility to quantify the cell number in various vessels including well plates, cultivation flasks, Petri dishes or even coverslips at reasonable cost
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
