---
title: "insane: Experimental Protocol"
author: "Amélie Bonnefond, Ph.D."
email: "amelie.bonnefond@cnrs.fr"
references:
- id: ndiaye_expression_2017
  title: Expression and functional assessment of candidate type 2 
    diabetes susceptibility genes identify four new genes contributing to human insulin secretion
  author:
      - family: Ndiaye
        given: Fatou K. 
      - family: Ortalli
        given: Ana 
      - family: Canouil
        given: Mickaël
      - family: Huyvaert
        given: Marlène
      - family: Salazar-Cardozo
        given: Clara
      - family: Lecoeur
        given: Cécile
      - family: Verbanck
        given: Marie
      - family: Pawlowski
        given: Valérie
      - family: Boutry
        given: Raphaël
      - family: Durand
        given: Emmanuelle
      - family: Rabearivelo
        given: Iandry
      - family: Sand
        given: Olivier
      - family: Marselli
        given: Lorella
      - family: Kerr-Conte
        given: Julie
      - family: Chandra
        given: Vikash
      - family: Scharfmann
        given: Raphaël
      - family: Poulain-Godefroy
        given: Odile
      - family: Marchetti
        given: Piero
      - family: Pattou
        given: François
      - family: Abderrahmani
        given: Amar
      - family: Froguel
        given: Philippe
      - family: Bonnefond
        given: Amélie
  container-title: Molecular Metabolism
  volume: 6
  issn: 2212-8778
  URL: 'http://dx.doi.org/10.1016/j.molmet.2017.03.011'
  DOI: 10.1016/j.molmet.2017.03.011
  issue: 6
  publisher: Molecular Metabolism
  page: 459-470
  type: article-journal
  issued:
    year: 2017
    month: 6
link-citations: true
nocite: | 
  @ndiaye_expression_2017
output: 
  rmarkdown::html_vignette:
    number_sections: false
    toc: false
    toc_depth: 3
    fig_width: 6.3
    fig_height: 4.7
vignette: >
  %\VignetteIndexEntry{insane: Experimental Protocol}
  %\VignetteEngine{knitr::rmarkdown}
  %\VignetteEncoding{UTF-8}
---

```{r setup, include = FALSE}
options(rmarkdown.html_vignette.check_title = FALSE)
knitr::opts_chunk$set(
  collapse = TRUE,
  comment = "#>",
  fig.path = "README-",
  out.width = "100%"
)
```

```{r logo, echo = FALSE, out.width = "150px", fig.align = "center"}
knitr::include_graphics(path = "insane.png")
```

### Experimental Protocol

#### Culture of EndoC-βH1 cells

EndoC-βH1 cells are cultured in Optiβ medium (Univercell). 
Cells are seeded at a density of 2.5&nbsp;×&nbsp;10<sup>6</sup> in T25 flasks coated with β-coat (Univercell) according to manufacturer’s instructions, and cultured at 37&nbsp;°C and 5&nbsp;% CO2. 

#### Transfection of siRNA into EndoC-βH1 cells

In case of transfection, siRNAs are transfected into EndoC-βH1 cells using Lipofectamine 2000 (Invitrogen).  
Freshly trypsinized EndoC-βH1 (0.5&nbsp;×&nbsp;10<sup>6</sup> cells) are incubated in suspension with Lipofectamine-siRNA complex in Opti-MEM (Invitrogen) containing 50&nbsp;μM 2-mercaptoethanol, 10&nbsp;mM nicotinamide (Calbiochem, Merck Millipore), 5.5&nbsp;mg/ml human transferrin (Sigma-Aldrich), 6.7&nbsp;ng/ml sodium selenite (Sigma-Aldrich), 100&nbsp;U/ml penicillin and 100&nbsp;mg/ml streptomycin (Life Technologies) for 10&nbsp;minutes and then are plated onto culture wells coated with β-oat (Univercell).  
Six hours later, the medium is replaced.  
We previously used ON-TARGETplus siRNA SMARTpool for each gene of interest (20-30&nbsp;nM) and ON-TARGETplus nontargeting pool for controls (siNTP; Dharmacon, Thermo Fisher Scientific, Waltham, MA, USA) [@ndiaye_expression_2017].  
Cells are analyzed 72 hours post transfection.

#### Assessment of insulin secretion from EndoC-βH1 cells

EndoC-βH1 cells seeded onto β-coat-coated 96-well plates at 5&nbsp;×&nbsp;10<sup>4</sup> cells/well are incubated overnight in no glucose Dulbecco’s Modified Eagle’s Medium (Sigma Aldrich) supplemented with 2&nbsp;% BSA Fraction V, 50&nbsp;μM 2-mercaptoethanol, 10&nbsp;mM nicotinamide (Calbiochem, Merck Millipore), 5.5&nbsp;mg/ml human transferrin (Sigma-Aldrich), 6.7&nbsp;ng/ml sodium selenite (Sigma-Aldrich), 100&nbsp;U/ml penicillin and 100&nbsp;mg/ml streptomycin (Life Technologies) and that contained 2.8&nbsp;mM glucose.  
The day after, they are incubated in HEPES-buffered Krebs-Ringer Buffer (KRB; 116&nbsp;mmol/l NaCl, 5.06&nbsp;mmol/l KCl, 1.007&nbsp;mmol/l CaCl2, 1.01&nbsp;mmol/l MgCl2, 23.96&nbsp;mmol/l NaHCO3, 10&nbsp;mmol/l HEPES, pH&nbsp;7.4, and 0.1&nbsp;% BSA solution) that contained 0.5&nbsp;mM glucose ±&nbsp;0.5 mM IBMX (Sigma Aldrich) for 60&nbsp;minutes at 37&nbsp;°C.  
This supernatant is subsequently collected (supernatant 1 [SN1]) and replaced with 16.7&nbsp;mM glucose KRB ±&nbsp;0.5 mM IBMX for a 60-minute incubation at 37&nbsp;°C and then collected (supernatant 2 [SN2]).  
For insulin content measurement, cells are lysed with TETG buffer (20&nbsp;mM Tris-HCl pH&nbsp;8.0, 137&nbsp;mM NaCl, 1&nbsp;% Triton X-100, 10&nbsp;% Glycerol, 2&nbsp;mM EGTA with protease inhibitors; Roche).  
Lysate, SN1 and SN2 are centrifuged for 5&nbsp;minutes at 700&nbsp;g.  
Samples are kept frozen at -20&nbsp;°C before use.  
Insulin concentration in the supernatants and intracellular content of the EndoC-βH1 cells are measured by ELISA according to manufacturer’s instructions using the Human Insulin Kit (Mercodia, Uppsala, Sweden).  
Briefly, 25&nbsp;µl of either blank, standard (from 0.2 to 6&nbsp;µg/ml), or diluted samples (1:400 for lysates and 1:16 for supernatants) and 100&nbsp;µl 1X conjugate enzyme are put in the 96-well plate coated with insulin antibody for 1&nbsp;hour under agitation at room temperature.  
The plate is then washed six times with wash buffer 1X solution.  
Then, 200&nbsp;µl substrate is added in the plate for a 15-minute incubation.  
Then, 50&nbsp;µl stop solution is added and the absorbance at 450&nbsp;nm is measured, using technical duplicates of experimental triplicates, leading to six measurements per experimental conditions (that are subsequently repeated).

*Note*: absorbance values need to be written within the Excel template provided by the **insane** package.

#### References
