Measurement of Secreted Embryonic Alkaline Phosphatase

Materials and Reagents

1. 96-well plate, transparent (Corning, catalog number: 3365)

2. 24-well plate, transparent (Corning, catalog number: 3537)

3. T25 flask (Corning, catalog number: 430639)

4. HEK-293T (ATCC, CRL-11268)

5. Dulbecco's modified Eagle's medium (DMEM) (Gibco, catalog number: 31600-083)

6. Fetal bovine serum (FBS) (Gibco, catalog number: 10270-106)

7. 0.25% trypsin-EDTA

8. 1% (vol/vol) penicillin and streptomycin solution (Beyotime Inc., catalog number: ST488-1/ST488-2)

9. p-nitrophenylphosphate (Sangon Biotech, CAS: 333338-18-4)

10. L-homoarginine (Sangon Biotech, CAS: 1483-01-8)

11. MgCl₂·6H₂O (Sangon Biotech, CAS: 7791-18-6)

12. Diethanolamine (Sangon Biotech, CAS: 111-42-2)

13. NaCl (Sangon Biotech, CAS: 7647-14-5)

14. KCl (Sangon Biotech, CAS: 7647-40-7)

15. Na₂HPO₄ (Sangon Biotech, CAS: 7558-79-4)

16. KH₂PO₄ (Sangon Biotech, CAS: 7778-77-0)

17. HCl (Sinopharm Chemical Reagent Co., Ltd., CAS: 7647-01-0）

18. Polyethyleneimine (PEI, molecular weight 40,000, stock solution 1 mg/mL in ddH₂O) (Polysciences, catalog number: 24765)

19. 1× PBS (see Recipes)

20. 2× SEAP assay buffer (see Recipes)

21. Substrate solution (see Recipes)

22. Plasmids (see Table 1)

    
    

    Table 1.Plasmids designed and used in this study

    Plasmid	Description and cloning strategy	Reference
    pXY137	Constitutive mammalian BldD and BphS expression vector (ITR-PhCMV-p65-VP64-BldD-pA-PhCMV-BphS-P2A-YhjH-pA-ITR)	Shao et al. (2017)
    pZQ5	Far-red light-inducible expression vector (2*crRNA binding site-PhCMVmin-SEAP-pA)	This work
    pZQ28	Constitutive crRNA2SEAP expression vector (PU6-crRNASEAP-pA)	This work
    pZQ113	Constitutive mammalian PhCMV-driven expression vector (PhCMV-scFv-45bp linker-p65-HSF1-pA)	This work
    pZQ116	Far-red light-inducible expression vector (PFRL2-dAsCas12a-NLS-10×GCN4-pA; PFRL3, (OwhiG)2-PhCMVmin)	This work

Equipment

1. Water bath

2. CO₂ incubator

3. Constant temperature incubator (65°C)

4. Synergy H1 hybrid multi-mode microplate reader (BioTek Instruments, Inc.)

5. Multipass pipette

6. Reagent reservoirs

7. Hemocytometer (Merck, catalog number: Z359629)

Software

1. Excel (Microsoft)

2. Gen5 software (version: 2.04)

Procedure

Cell culture

1. Warm DMEM supplemented with 10% (vol/vol) FBS and 1% (vol/vol) penicillin and streptomycin solution (hereafter referred to as complete DMEM) and 1× PBS in a 37°C water bath for at least 15 min.

2. Wash ~90% confluent human embryonic kidney cells (HEK-293T) in a T25 flask with 3 mL of 1× PBS, aspirate the PBS, and add 0.5 mL of 0.25% trypsin-EDTA.

3. Incubate the cells at 37°C in a 5% CO₂ incubator for 3–5 min, add 3 mL of prewarmed complete DMEM, and transfer the cell suspensions to a 15 mL sterile conical tube.

4. Spin down the cells at 300 × g for 3 min at room temperature, discard the supernatant, and resuspend the cell pellet in 15 mL of complete DMEM.

5. Dispense 4 mL of the cell suspension into a T25 flask and incubate it for three days at 37°C in a humidified atmosphere containing 5% CO₂ before subculturing again.

Cell transfection

1. The 293T cell suspension should be prepared (as described in the Cell culture procedure) the day before transfection (Day 1) (Figure 1).

   
   
   Figure 1. Schematic diagram of the schedule for cell transfection and SEAP analysis

   
   

2. Count the cells using a hemocytometer and seed 6 × 10⁴ HEK-293T cells per well in a 24-well cell culture plate; culture for 18 h.

3. The next day (Day 2), prepare the transfection mix as follows:

   1. Add 500 ng of the corresponding plasmid (see Table 1) into 50 μL of FBS-free and antibiotic-free DMEM medium for each well of a 24-well plate.

   2. Add 1.5 μL of PEI (1 μg/μL, PEI and DNA at a ratio of 3:1) into the above plasmid DNA solution.

   3. To scale up, multiply the quantities by the number of replicates.

4. Vortex vigorously for 30 s.

5. Incubate the DNA-PEI mixture solution at room temperature for 15 min.

6. Add 50 μL of transfection mix dropwise to each well. Rock the slide gently a few times and incubate at 37°C in a humidified atmosphere containing 5% CO₂.

7. Six hours after transfection, change the medium with fresh complete DMEM and incubate the 24-well plate at 37°C in a 5% CO₂ incubator until Day 3.

8. On Day 3, the transfected slides can be used to perform SEAP reporter assay.

SEAP reporter assay

1. Aspirate 200 μL of cell culture supernatant from each sample and transfer into a chamber of a 96-well plate.

2. Heat-inactivate the cell culture supernatants at 65°C for 30 min.

3. Prewarm 2× SEAP assay buffer (see Recipes) at 37°C and protect from light.

4. Mix 100 μL of 2× SEAP buffer with 20 μL of substrate solution (see Recipes) in the dark.

5. Add 120 μL of the above-mentioned mixed substrate solution into 80 μL of heat-inactivated cell culture supernatant. At least three replicates should be performed for each group. The media will change color from pink to orange in the presence of SEAP (Figure 2).

   
   
   Figure 2. Detection of SEAP production at the indicated time points after illumination. In the presence of SEAP, the media changes from pink to orange. The yellow color intensity correlates with light illumination time.

   
   

6. Measure light absorbance at 405 nm at 37°C for 15 min using a multi-mode microplate reader. See Figure 3 for the detailed procedure.

   
   

   
   Figure 3. Schematic diagram of SEAP reporter assay

   
   

7. Calculate the levels of SEAP activity using data points that yield a rate of change in light absorbance that is linear with respect to time. (If available, a computer-linked kinetic ELISA plate reader is most convenient, as this will precisely calculate the linear change in A₄₀₅ in all 96 wells of a microplate over time.)

Data analysis

For each illumination time, data are expressed as mean ± standard deviation (SD); n = 3 independent experiments. The data can also be represented as fold change difference compared to non-stimulated cells and analyzed by one-way ANOVA followed by Tukey’s post-hoc tests. Perform statistical analysis using GraphPad Prism or other software.

Figure 4. Quantification of far-red light-inducible SEAP expression kinetics. Transfected HEK-293T cells were illuminated with far-red light for different time periods (0–120 min). SEAP production in the culture supernatant was profiled at 24 h after illumination. **P < 0.01, ***P < 0.001 illumination groups vs. 0 min.

Notes

1. The mixture of 2× SEAP buffer and substrate solution should be protected from light, quickly added to cell culture supernatants, and detected immediately.

2. If SEAP production is too high, the samples should be diluted with PBS buffer solution.

Recipes

1. 1× PBS buffer

   Reagent	Final concentration	Amount
   NaCl	137 mM	8.00 g
   KCl	2.7 mM	0.20 g
   Na2HPO4	10 mM	1.42 g
   KH2PO4	1.8 mM	0.24 g
   H2O	n/a	800 mL
   Adjust pH to 7.4 with HCl, then add ddH2O to final volume (1,000 mL)
   Total	n/a	1,000 mL




2. 2× SEAP buffer

   Reagent	Final concentration	Amount
   L-homoarginine	20 mM	4.4938 g
   MgCl2 (3 M)	1 mM	0.334 mL
   Diethanolamine	21% (v/v)	210 mL
   Adjust pH to 9.8 with HCl, then add dH2O to final volume (1,000 mL)
   Total	n/a	1,000 mL

   Store at 4°C and protect from light.




3. Substrate solution

   Reagent	Final concentration	Amount
   p-nitrophenylphosphate	120 mM	2.226 g
   2× SEAP buffer	n/a	50 mL
   Total	n/a	50 mL

   Aliquot, store at -20°C, and protect from light.

Acknowledgments

This protocol was adapted from previous work (Wang et al., 2021. DOI: 10.1126/sciadv.abh2358). This work was financially supported by grants from the National Key R&D Program of China (No. 2019YFA0904500, No. 2019YFA0110802), the National Natural Science Foundation of China (NSFC: No. 32171414, No. 31971346, No. 31861143016, No. 31870861), the Nature Science Foundation of Chongqing, China (No. CSTB2022NSCQ-MSX0461), the Open Project Program of State Key Laboratory of Dairy Biotechnology (No. SKLDB2021-001), and the Instruments Sharing Platform of the School of Life Sciences, East China Normal University.

Competing interests

All authors wish to declare no competing interests.

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