Electrogenetic cellular insulin release for real-time glycemic control in type 1 diabetic mice

Data underlying the figures in the publication “Electrogenetic cellular insulin release for real-time glycemic control in type 1 diabetic mice”, published in Science, 2020, 368, 993-1001. https://science.sciencemag.org/content/368/6494/993

Table of contents:

1. Electrogenetics source data file; File openable with Graphpad Prism containing the source data for the main Figures: 1-4 (Transgene expression by SEAP measurement), 5b, 6a, 6f (Insulin by ELISA), 5c, 6b, 6e (NanoLuc luciferase), 7c (NanoLuc luciferase in vivo) and 7a, 7d (Glycemia).

Transgene expression by SEAP measurement

Data for main Figures 1, 2, 3, 4.

SEAP (human placental secreted alkaline phosphatase) levels were profiled in cell culture supernatants using a colorimetric assay. 100 µL 2x SEAP assay buffer (20 mM homoarginine, 1 mM MgCl2, 21% diethanolamine, pH 9.8) was mixed with 80 µL heat-inactivated (30 min at 65°C) cell culture supernatant. After the addition of 20 µL substrate solution (120 mM p-nitrophenyl phosphate; cat. no. AC128860100, Thermo Fisher Scientific), the absorbance time course was recorded for 45 min at 405 nm and 37°C using a Tecan Genios PRO plate reader (cat. no. P97084; Tecan Group AG, Maennedorf, Switzerland) and the SEAP levels were determined as follows: first, absorbance change over time (slope) was calculated. According to the Beer–Lambert’s law, absorbance is proportional to the concentration of a colored compound and depends on the light path length (d) and extinction coefficient (ε) (ε for p-nitrophenyl (εpNP) = 18.600 M−1 cm−1). Enzymatic activity EA [U/L] was calculated from the equation: EA = slope × dilution factor × εpNP−1 × d−1 

Values in the file present determined SEAP levels.

Insulin by ELISA

Data for Figures 5b, 6a, 6f.

Values in the file present Insulin level as determined by ELISA kit. The assay was performed according to manufacturer’s instructions.

NanoLuc luciferase

Data for Figures 5c, 6b, 6e.

NanoLuc® luciferase was quantified in cell culture supernatants using the Nano-Glo® Luciferase Assay System (cat. no. N1110; Promega, Duebendorf, Switzerland). In brief, 7.5 µL of cell culture supernatant was added per well of a black 384-well plate and mixed with 7.5 µL substrate-containing assay buffer. Total luminescence was quantified using a Tecan Genios PRO plate reader (Tecan Group AG).

Values in the file present measured luminescence levels.

NanoLuc luciferase in vivo

Data for Figure 7c.

Aliquots of 15 µL of whole-blood samples were diluted in 5 μL of 50 mM EDTA and frozen at -20 °C until NanoLuc® quantification as described above.

Values in the file present measured luminescence levels normalized to time point 0 (Normalization individually for each mouse).

Glycemia

Data for Figures 7a, 7d.

Blood glucose level was determined using a glucometer (Contour®Next, Bayer Healthcare, Leverkusen, Germany).

Values in the file present measured glucose levels.

2. Figure 7b; Data for Figure 7b. File openable with Graphpad Prism.

Blood glucose level was determined using a glucometer (Contour®Next, Bayer Healthcare, Leverkusen, Germany).

Values in the file present measured glucose levels.

3. Figure 6c and 6d; Excel file with the data for Figures 6c, 6d.

NanoLuc® luciferase was quantified in cell culture supernatants using the Nano-Glo® Luciferase Assay System (cat. no. N1110; Promega, Duebendorf, Switzerland). In brief, 7.5 µL of cell culture supernatant was added per well of a black 384-well plate and mixed with 7.5 µL substrate-containing assay buffer. Total luminescence was quantified using a Tecan Genios PRO plate reader (Tecan Group AG).

Values in the file present measured luminescence levels.

本数据集对应发表于《Science》2020年，第368卷，第993-1001页的论文《Electrogenetic cellular insulin release for real-time glycemic control in type 1 diabetic mice》的配图原始数据，论文链接：https://science.sciencemag.org/content/368/6494/993

目录：

1. 电遗传学原始数据文件；可通过Graphpad Prism打开，包含主图1-4（基于SEAP检测的转基因表达）、5b、6a、6f（ELISA检测胰岛素）、5c、6b、6e（NanoLuc荧光素酶（NanoLuc® luciferase））、7c（体内NanoLuc荧光素酶）以及7a、7d（血糖水平）的原始数据。

### 基于SEAP检测的转基因表达
对应主图1、2、3、4的原始数据。
采用比色法对细胞培养上清中的SEAP（人胎盘分泌型碱性磷酸酶，human placental secreted alkaline phosphatase）水平进行检测。取100 μL 2× SEAP检测缓冲液（20 mM 高精氨酸、1 mM 氯化镁、21% 二乙醇胺，pH 9.8），与80 μL 经65℃热灭活30分钟的细胞培养上清混合。加入20 μL 底物溶液（120 mM 对硝基苯磷酸；货号AC128860100，赛默飞世尔科技（Thermo Fisher Scientific））后，使用Tecan Genios PRO读板仪（货号P97084；特康集团（Tecan Group AG），梅嫩多夫，瑞士）于405 nm波长、37℃下记录45分钟的吸光度时间进程。SEAP水平计算方式如下：首先计算吸光度随时间的变化速率（斜率）。根据比尔-朗伯定律（Beer–Lambert’s law），吸光度与有色化合物浓度成正比，且取决于光程长度（d）和消光系数（ε），其中对硝基苯（εpNP）的消光系数εpNP = 18600 M⁻¹·cm⁻¹。酶活性EA [U/L] 通过以下公式计算：EA = 斜率 × 稀释因子 × εpNP⁻¹ × d⁻¹。
文件中的数值为测定得到的SEAP水平。

### ELISA检测胰岛素
对应图5b、6a、6f的原始数据。
文件中的数值为通过酶联免疫吸附测定（ELISA）试剂盒测定得到的胰岛素水平。检测操作严格按照试剂盒制造商的说明书进行。

### NanoLuc荧光素酶（NanoLuc® luciferase）检测
对应图5c、6b、6e的原始数据。
使用Nano-Glo®荧光素酶检测系统（货号N1110；普洛麦格（Promega），迪本多夫，瑞士）对细胞培养上清中的NanoLuc荧光素酶进行定量。具体操作简述如下：向黑色384孔板的每孔中加入7.5 μL 细胞培养上清，再加入7.5 μL 含底物的检测缓冲液混匀。使用Tecan Genios PRO读板仪（特康集团）对总发光强度进行定量。
文件中的数值为测定得到的发光强度水平。

### 体内NanoLuc荧光素酶检测
对应图7c的原始数据。
取15 μL 全血样本，用5 μL 50 mM EDTA 稀释后于-20℃冻存，直至按前述方法进行NanoLuc荧光素酶定量。
文件中的数值为以时间点0为基准进行标准化后的发光强度水平（每只小鼠单独进行标准化）。

### 血糖水平检测
对应图7a、7d的原始数据。
使用血糖仪（Contour®Next，拜耳医疗（Bayer Healthcare），勒沃库森，德国）测定血糖水平。
文件中的数值为测定得到的葡萄糖浓度水平。

2. 图7b原始数据；对应图7b的原始数据。可通过Graphpad Prism打开。
使用血糖仪（Contour®Next，拜耳医疗，勒沃库森，德国）测定血糖水平。
文件中的数值为测定得到的葡萄糖浓度水平。

3. 图6c和6d原始数据；包含图6c、6d的原始数据的Excel文件。
使用Nano-Glo®荧光素酶检测系统（货号N1110；普洛麦格，迪本多夫，瑞士）对细胞培养上清中的NanoLuc荧光素酶进行定量。具体操作简述如下：向黑色384孔板的每孔中加入7.5 μL 细胞培养上清，再加入7.5 μL 含底物的检测缓冲液混匀。使用Tecan Genios PRO读板仪（特康集团）对总发光强度进行定量。
文件中的数值为测定得到的发光强度水平。