Objective      To determine the influential factors of reactive oxidative species (ROS) and cell nucleus co-staining with 2′

7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and 4′

6′-diamidino-2-phenylindole (DAPI).       Methods      RAW264.7 cells were fixed with paraformaldehyde (0%

0.5%

1%

2% and 4%) for 10  and 30 min

and 1

2 and 4 h. Then the ROS and DAPI were detected and th

e fluorescent signals were analyzed by ImageXpress XLS. Meanwhile

another fixing reagent

ethanol

was adopted to do the similar experiment

and ROS was detected before or after the cells were fixed for 20 min. Thereafter

DAPI staining was performed at 12

4 and 1 h

20 and 0 min before DCFH-DA incubation

and the fluorescent signals were detected via the same technology. In addition

LPS and H

2

O

2

were used to induce ROS

and DAPI was stained in advance. Twelve h later

the number of fluorescent cells and the average fluorescence intensity of ROS were detected. All data was analyzed by t-test via SPSS18.0 software.       Results      No matter which factors of cell fixation (fixing concentrations

fixing reagents and fixing sequences) were adopted

the fluorescent intensity of ROS was decreased while the fluorescent signals of DAPI was increased. Along with the dying time

the strength and uniformity of DAPI staining became better without fixation. ROS was detected after DAPI staining for 12 h

and there was no significant influence on the number of fluorescent cells and average fluorescence intensity of ROS (P0.05).       Conclusion      It is not suggested to carry out DCFH-DA incubation and cell fixation simultaneously to detect the fluorescence of ROS. However

the measuring effect of fluorescence intensity will not be impacted significantly if DAPI staining is accomplished at 12 h before DCFH-DA incubation. Thus

this proposal may obtain the optimal effect of ROS and DAPI co-staining.