Figure1.Determination of apoptosis in transiently transfected murine [beta] tumor cells. (A) The number of apoptotic [beta]HC 13T tumor cells (% apoptosis) in the total transfected eGFP-positive population is shown. Co-transfections of pEGFP and the plasmids as indicated were carried out using LipofectAMINE as described in the text. Apoptotic cells were measured by flow cytometric analysis either using PI staining and determination of sub-2 N DNA content (GFP/PI; black bars) or by the incorporation of fluorescent Cy5-CTP during the TdT reaction (GFP/TUNEL; white bars). Staurosporine (800 ng/ml) was added following transfection as indicated (+ staurosporine). Excitation wavelength of 488 nm was used for eGFP and PI, 647 nm for Cy5 and multiline UV for DAPI. Emmision fluorescence was collected using a 530/20 nm bandpass filter for eGFP, 610 nm longpass filter for PI, 675/20 nm bandpass filter for Cy5 and a 424/44 nm bandpass filter for DAPI. Doublets were excluded by pulse processing. Cells expressing eGFP were gated and analysed for either Cy5- or PI-fluorescence. Data were analysed using CELLQuest software (Becton Dickinson). Each bar represents the average of three transfections, standard deviations are indicated by error bars. Each measurement included 40 000 total events gated for size and single cells. Transfection efficiency was 20-30%. (B) The normalised percentage of apoptosis is shown for the various constructs. The apoptotic index was normalised using the function (% apopotosis in X / % apoptosis in eGFP-C1/E1A) × 100. The apoptotic index was normalised for each detection method used and for each post-transfection treatment employed (i.e. +/- staurosporine). Note the pro-apoptotic and anti-apoptotic effect of E1A and E1B-19K, respectively.
To demonstrate the general usefulness of our assay, the GFP/PI method was tested on a non-transformed rat fibroblast cell line (Rat1A) which is less sensitive to apoptotic stimuli. Rat1A cells were transiently transfected using either LipofectAMINE as described for the [beta] tumor cells above, or polyethylenimine-(PEI 2000)-DNA-adenovirus complexes as described (24). A comparison of the different transfection and apoptotic detection methods using Rat1A cells is presented in Table1. As observed with the [beta] tumor cell lines, the GFP/PI method is a reliable apoptotic assay for transiently transfected Rat1A cells (Table1) as well as for a human lung carcinoma cell line A549 (ATCC CCL-185) (24).
aTransfection of Rat1A cells with pEGFP-C1 and the constructs as indicated using either LipofectAMINE Reagent or PEI(2000)-DNA-adenovirus complexes. Following transfection the cells were treated with staurosporine (800 ng/ml) for 16 h. The apoptotic index of the transfected cells was analysed by flow cytometry as described in the text and in the legend to Figure 1. Each value represents the average of three transfections, standard deviation is indicated (s.d.). Transfection efficiency was 25-30% both using LipofectAMINE and PEI/Adeno. bThe apoptotic index of eGFP-positive cells was determined by PI staining and sub-2 N DNA content. cThe apoptotic index of eGFP-positive cells was determined by the incorporation of fluorescent Cy5-CTP during the TUNEL reaction. dThe normalised apoptotic index was calculated as described in the legend to Figure 1.
The PFA/EtOH fixation procedure used in our protocol is essential. Omission of the mild PFA fixation step leads to the rapid leakage of eGFP from the cell and impairs detection of transiently transfected cells. The EtOH fixation/permeabilisation step allows diffusion of low molecular weight DNA products from apoptotic cells and is required to detect the characteristic sub-2 N DNA peak upon PI staining (6,17).
In conclusion, we present a rapid, efficient and reproducible method for determining the apoptotic potential of a given gene of interest in a variety of cell lines. Our method is applicable to and compatible with a variety of cell types and transfection methods. It combines the ease of using GFP as a marker for transfection and sub-2 N PI staining as a simple, inexpensive assay for apoptosis. Furthermore, this assay can be carried out on a relatively simple FACScan analyser and does not require elaborate FACS sorter technology or expertise.
ACKNOWLEDGEMENTS
The authors wish to thank Johannes Hoffman for providing the TdT/Cy5-CTP protocol for FACS analyses, Eileen White for pCMV-E1A and pCMV-E1B-19K and Thomas Bader and Kanaga Sabapathy for critical reading of the manuscript. This work was supported in part by the Austrian Industrial Promotion Fund.
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