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| We used the AttoPhos® AP Fluorescent Substrate System (Cat.# S1000) and Western Blue® Stabilized Substrate for Alkaline Phosphatase (Cat.# S3841) to detect alkaline phosphatase activity in transfected NIH3T3 cells in situ. Permeabilization of the cells was not required but Triton® X-100 treatment increased the signal. |
Introduction
AttoPhos® AP Fluorescent Substrate System is recommended for use as a fluorescent substrate of alkaline phosphatase (AP) in ELISA format, while Western Blue® Stabilized Substrate is recommended for use as a colorimetric substrate for AP detection in membrane-bound applications (i.e., Western blots). In this study, both substrates were used as in situ detection reagents for AP in fixed NIH3T3 cells. NIH3T3 cells were transiently transfected with pLAPSN containing the AP gene, leading to the cytoplasmic accumulation of AP as a reporter molecule. After a suitable expression time, the cells were fixed with 4% paraformaldehyde and permeabilized under different conditions. AttoPhos® Substrate and Western Blue® Substrate were then applied, and cells were viewed using fluorescence (FITC filter) and phase microscopy.
Procedures
Transfection of NIH3T3 cells: NIH3T3 cells were grown to 70% confluency in a 24 well tissue culture plate. pLAPSN DNA (25µg) was added to 5ml serum-free DMEM. A control reaction without DNA was also prepared. The DNA/DMEM solution was combined with 75µl of TransFast™ Transfection Reagent* (Cat.# E2431), and incubated at room temperature for 10 minutes. After aspirating the medium from the 24 well plate, 200µl of either DNA/lipid mixture or the control mixture were added to the appropriate wells and the cells were incubated at 37°C/10% CO2 for 60 minutes. The transfected cells were overlaid with 1ml of DMEM + 10% calf serum, and expression of AP was assayed 48 hours post-transfection.
Detection of AP activity with AttoPhos® Substrate and Western Blue® Substrate: After two washes with PBS, the cells were fixed with 4% paraformaldehyde for 15 minutes and washed once with PBS. The cells were permeabilized with either 0.1% or 0.04% Triton® X-100 in PBS for 15 minutes. One column received no Triton® X-100. The Triton® X-100 was removed with a single PBS wash. The cells were treated with either AttoPhos® Substrate or Western Blue® Substrate (full strength) for 30 minutes on a shaking platform (gentle shaking). The cells were washed twice with PBS, covered with Vectashield® (Vector Labs) and DAPI, then viewed by fluorescence/phase microscopy.
Results
AttoPhos® Substrate appeared to react with expressed cytoplasmic AP in the NIH3T3 cells leading to green fluorescence in approximately 25% of the cells. Western Blue® Substrate appeared to react with the expressed cytoplasmic AP leading to a dark blue to black precipitate in approximately 25% of the cells. Reaction product for either substrate was not seen in all cells and did not appear in any nontransfected control cells, showing their specificity for the transiently expressed AP. The relative strength of permeabilization did have an obvious effect, which was more noticeable in Western Blue® Substrate-treated cells.
For the Western Blue® Substrate, cells that were not permeabilized showed a weak but discernible signal above nontransfected cells (Figure 1, Panel A). Cells treated with 0.04% Triton® X-100 showed a stronger signal, but a precipitate appeared outside the cells, which was apparently attached to the surface of the plate, since it did not wash away (Figure 1, Panel B). This background became much more intense with a 0.1% Triton® X-100 treatment, leading to difficulty in determining which cells were positive (Figure 1, Panel C). The increase in background with increased permeabilization is perhaps caused by the precipitate “leaking” back out of the cell and depositing on the plate surface.
For the AttoPhos® Substrate, reaction product was visible above background in the untreated nonpermeabilized cells (Figure 2, Panel A). However, the strongest signal was seen in the 0.1% Triton® X-100 permeabilized cells (Figure 2, Panel B).
AttoPhos® Substrate |
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Figure 2. Cells treated with AttoPhos® Substrate that were not permeabilized (Panel A) or permeabilized with 0.1% Triton® X-100 (Panel B). |
Conclusion
AttoPhos® Substrate and Western Blue® Substrate provide an easy and quick way to determine transfection efficiency in NIH3T3 cells expressing AP. Optimal conditions for use of this reagent as an in situ marker for AP may need to be refined for each cell type. Permeabilization was not required to give positive results with this cell line using Western Blue® or AttoPhos® Substrates, but increased signal was seen with the AttoPhos® Substrate following treatment with 0.1% Triton® X-100. Other lines may behave differently. We recommend that results from a short, weak permeabilization treatment be compared to results from no permeabilization.
