Hackuarium - User contributions [en]

Tumeric

2017-05-19T13:19:37Z

Bastien: /* Gallery */

Experiment 7avr17 

Aim:
To get a cell staining protocol that uses non-toxic (kitchen) ingredients 

To see if tumeric might stain cells.

1) dry down some cheek cells on a slide, to fix with EtOH

2) make solution of Tumeric in ethanol (doesn't seem to dissolve in saline) 

3) put a drop of tumeric solution on one patch, methylene blue on another and compare 

Is any color visible? 
What about after treatments for comet tests?

Anna and Bastien already counted cheek cells, and are making pads before RA joined in.
Had plan for an experiment with the osmotic shock and the detergent treatment already.
will try staining one of each with MeBlue and one with tumeric?

The tumeric did not seem to do much, if anything...

=== Gallery ===

<gallery mode=packed widths=300 heights=300>
File:MeBlueControl_170417.jpg|Methylene Blue positive control for the tumeric experiment
File:TumericAttempt_170417.jpg|Cells incubated with tumeric extracts. No real staining nor specificity with the staining.
</gallery>

===Links===

[https://drive.google.com/file/d/0B1jaPHOkH_42WVRfLXJ6ZWZnWVE/view?usp=sharing Picture of cells incubated in tumeric extract]

[https://drive.google.com/file/d/0B1jaPHOkH_42aUJydUdoWVZOOVE/view?usp=sharing Methylene Blue control]

Tumeric

2017-05-19T13:16:22Z

Bastien: /* Galery */

Experiment 7avr17 

Aim:
To get a cell staining protocol that uses non-toxic (kitchen) ingredients 

To see if tumeric might stain cells.

1) dry down some cheek cells on a slide, to fix with EtOH

2) make solution of Tumeric in ethanol (doesn't seem to dissolve in saline) 

3) put a drop of tumeric solution on one patch, methylene blue on another and compare 

Is any color visible? 
What about after treatments for comet tests?

Anna and Bastien already counted cheek cells, and are making pads before RA joined in.
Had plan for an experiment with the osmotic shock and the detergent treatment already.
will try staining one of each with MeBlue and one with tumeric?

The tumeric did not seem to do much, if anything...

=== Gallery ===

<gallery mode=packed>
[[File:MeBlueControl_170417.jpg|225px|300px|Methylene Blue positive control for the tumeric experiment]]
[[File:TumericAttempt_170417.jpg|311px|300px|Cells incubated with tumeric extracts. No real staining nor specificity with the staining.]]
</gallery>

===Links===

[https://drive.google.com/file/d/0B1jaPHOkH_42WVRfLXJ6ZWZnWVE/view?usp=sharing Picture of cells incubated in tumeric extract]

[https://drive.google.com/file/d/0B1jaPHOkH_42aUJydUdoWVZOOVE/view?usp=sharing Methylene Blue control]

Tumeric

2017-05-19T13:03:31Z

Bastien:

Experiment 7avr17 

Aim:
To get a cell staining protocol that uses non-toxic (kitchen) ingredients 

To see if tumeric might stain cells.

1) dry down some cheek cells on a slide, to fix with EtOH

2) make solution of Tumeric in ethanol (doesn't seem to dissolve in saline) 

3) put a drop of tumeric solution on one patch, methylene blue on another and compare 

Is any color visible? 
What about after treatments for comet tests?

Anna and Bastien already counted cheek cells, and are making pads before RA joined in.
Had plan for an experiment with the osmotic shock and the detergent treatment already.
will try staining one of each with MeBlue and one with tumeric?

The tumeric did not seem to do much, if anything...

=== Galery ===

{{Gallery
|title=Cultural depictions of George Washington
|width=300 | height=300
|align=center
File:MeBlueControl_170417.jpg|Methylene Blue positive control for the tumeric experiment
File:TumericAttempt_170417.jpg|Cells incubated with tumeric extracts. No real staining nor specificity with the staining.
}}

===Links===

[https://drive.google.com/file/d/0B1jaPHOkH_42WVRfLXJ6ZWZnWVE/view?usp=sharing Picture of cells incubated in tumeric extract]

[https://drive.google.com/file/d/0B1jaPHOkH_42aUJydUdoWVZOOVE/view?usp=sharing Methylene Blue control]

Tumeric

2017-05-19T13:01:05Z

Bastien: /* Galery */

Experiment 7avr17 

Aim:
To get a cell staining protocol that uses non-toxic (kitchen) ingredients 

To see if tumeric might stain cells.

1) dry down some cheek cells on a slide, to fix with EtOH

2) make solution of Tumeric in ethanol (doesn't seem to dissolve in saline) 

3) put a drop of tumeric solution on one patch, methylene blue on another and compare 

Is any color visible? 
What about after treatments for comet tests?

Anna and Bastien already counted cheek cells, and are making pads before RA joined in.
Had plan for an experiment with the osmotic shock and the detergent treatment already.
will try staining one of each with MeBlue and one with tumeric?

The tumeric did not seem to do much, if anything...

=== Galery ===

<gallery>
File:MeBlueControl_170417.jpg|Methylene Blue positive control for the tumeric experiment
File:TumericAttempt_170417.jpg|Cells incubated with tumeric extracts. No real staining nor specificity with the staining.
</gallery>

===Links===

[https://drive.google.com/file/d/0B1jaPHOkH_42WVRfLXJ6ZWZnWVE/view?usp=sharing Picture of cells incubated in tumeric extract]

[https://drive.google.com/file/d/0B1jaPHOkH_42aUJydUdoWVZOOVE/view?usp=sharing Methylene Blue control]

Tumeric

2017-05-19T13:00:01Z

Bastien: /* Galery */

Experiment 7avr17 

Aim:
To get a cell staining protocol that uses non-toxic (kitchen) ingredients 

To see if tumeric might stain cells.

1) dry down some cheek cells on a slide, to fix with EtOH

2) make solution of Tumeric in ethanol (doesn't seem to dissolve in saline) 

3) put a drop of tumeric solution on one patch, methylene blue on another and compare 

Is any color visible? 
What about after treatments for comet tests?

Anna and Bastien already counted cheek cells, and are making pads before RA joined in.
Had plan for an experiment with the osmotic shock and the detergent treatment already.
will try staining one of each with MeBlue and one with tumeric?

The tumeric did not seem to do much, if anything...

=== Galery ===

<gallery>
[[File:MeBlueControl_170417.jpg|thumbnail|left|225px|300px|Methylene Blue positive control for the tumeric experiment]]
[[File:TumericAttempt_170417.jpg|thumbnail|center|311px|300px|Cells incubated with tumeric extracts. No real staining nor specificity with the staining.]]
 
</gallery>

===Links===

[https://drive.google.com/file/d/0B1jaPHOkH_42WVRfLXJ6ZWZnWVE/view?usp=sharing Picture of cells incubated in tumeric extract]

[https://drive.google.com/file/d/0B1jaPHOkH_42aUJydUdoWVZOOVE/view?usp=sharing Methylene Blue control]

Tumeric

2017-05-19T12:58:35Z

Bastien:

Experiment 7avr17 

Aim:
To get a cell staining protocol that uses non-toxic (kitchen) ingredients 

To see if tumeric might stain cells.

1) dry down some cheek cells on a slide, to fix with EtOH

2) make solution of Tumeric in ethanol (doesn't seem to dissolve in saline) 

3) put a drop of tumeric solution on one patch, methylene blue on another and compare 

Is any color visible? 
What about after treatments for comet tests?

Anna and Bastien already counted cheek cells, and are making pads before RA joined in.
Had plan for an experiment with the osmotic shock and the detergent treatment already.
will try staining one of each with MeBlue and one with tumeric?

The tumeric did not seem to do much, if anything...

=== Galery ===

[[File:MeBlueControl_170417.jpg|thumbnail|left|225px|300px|Methylene Blue positive control for the tumeric experiment]]
[[File:TumericAttempt_170417.jpg|thumbnail|center|311px|300px|Cells incubated with tumeric extracts. No real staining nor specificity with the staining.]]
 
===Links===

[https://drive.google.com/file/d/0B1jaPHOkH_42WVRfLXJ6ZWZnWVE/view?usp=sharing Picture of cells incubated in tumeric extract]

[https://drive.google.com/file/d/0B1jaPHOkH_42aUJydUdoWVZOOVE/view?usp=sharing Methylene Blue control]

File:TumericAttempt 170417.jpg

2017-05-19T12:41:27Z

Bastien: Attempt to satin cells with Tumeric extract. Tumeric experiment, 17.04.17

Attempt to satin cells with Tumeric extract.

Tumeric experiment, 17.04.17

Tumeric

2017-05-19T12:39:46Z

Bastien: /* Galery */

Experiment 7avr17 

Aim:
To get a cell staining protocol that uses non-toxic (kitchen) ingredients 

To see if tumeric might stain cells.

1) dry down some cheek cells on a slide, to fix with EtOH

2) make solution of Tumeric in ethanol (doesn't seem to dissolve in saline) 

3) put a drop of tumeric solution on one patch, methylene blue on another and compare 

Is any color visible? 
What about after treatments for comet tests?

Anna and Bastien already counted cheek cells, and are making pads before RA joined in.
Had plan for an experiment with the osmotic shock and the detergent treatment already.
will try staining one of each with MeBlue and one with tumeric?

The tumeric did not seem to do much, if anything...

=== Galery ===

[[File:MeBlueControl_170417.jpg |225px|300px]]

[https://drive.google.com/file/d/0B1jaPHOkH_42WVRfLXJ6ZWZnWVE/view?usp=sharing Picture of cells incubated in tumeric extract]

[https://drive.google.com/file/d/0B1jaPHOkH_42aUJydUdoWVZOOVE/view?usp=sharing Methylene Blue control]

Tumeric

2017-05-19T12:31:51Z

Bastien: /* Galery */

Experiment 7avr17 

Aim:
To get a cell staining protocol that uses non-toxic (kitchen) ingredients 

To see if tumeric might stain cells.

1) dry down some cheek cells on a slide, to fix with EtOH

2) make solution of Tumeric in ethanol (doesn't seem to dissolve in saline) 

3) put a drop of tumeric solution on one patch, methylene blue on another and compare 

Is any color visible? 
What about after treatments for comet tests?

Anna and Bastien already counted cheek cells, and are making pads before RA joined in.
Had plan for an experiment with the osmotic shock and the detergent treatment already.
will try staining one of each with MeBlue and one with tumeric?

The tumeric did not seem to do much, if anything...

=== Galery ===

[[File:MeBlueControl_170417.jpg]]

[https://drive.google.com/file/d/0B1jaPHOkH_42WVRfLXJ6ZWZnWVE/view?usp=sharing Picture of cells incubated in tumeric extract]

[https://drive.google.com/file/d/0B1jaPHOkH_42aUJydUdoWVZOOVE/view?usp=sharing Methylene Blue control]

Tumeric

2017-05-19T12:31:19Z

Bastien:

Experiment 7avr17 

Aim:
To get a cell staining protocol that uses non-toxic (kitchen) ingredients 

To see if tumeric might stain cells.

1) dry down some cheek cells on a slide, to fix with EtOH

2) make solution of Tumeric in ethanol (doesn't seem to dissolve in saline) 

3) put a drop of tumeric solution on one patch, methylene blue on another and compare 

Is any color visible? 
What about after treatments for comet tests?

Anna and Bastien already counted cheek cells, and are making pads before RA joined in.
Had plan for an experiment with the osmotic shock and the detergent treatment already.
will try staining one of each with MeBlue and one with tumeric?

The tumeric did not seem to do much, if anything...

=== Galery ===

[[File:MetBlue MeBlueControl 170417.jpg]]

[https://drive.google.com/file/d/0B1jaPHOkH_42WVRfLXJ6ZWZnWVE/view?usp=sharing Picture of cells incubated in tumeric extract]

[https://drive.google.com/file/d/0B1jaPHOkH_42aUJydUdoWVZOOVE/view?usp=sharing Methylene Blue control]

Tumeric

2017-05-19T12:30:23Z

Bastien: /* Galery */

Experiment 7avr17 

Aim:
To get a cell staining protocol that uses non-toxic (kitchen) ingredients 

To see if tumeric might stain cells.

1) dry down some cheek cells on a slide, to fix with EtOH

2) make solution of Tumeric in ethanol (doesn't seem to dissolve in saline) 

3) put a drop of tumeric solution on one patch, methylene blue on another and compare 

Is any color visible? 
What about after treatments for comet tests?

Anna and Bastien already counted cheek cells, and are making pads before RA joined in.
Had plan for an experiment with the osmotic shock and the detergent treatment already.
will try staining one of each with MeBlue and one with tumeric?

The tumeric did not seem to do much, if anything...

=== Galery ===

[[File:MetBlue MeBlueControl_170417.jpg]]

[https://drive.google.com/file/d/0B1jaPHOkH_42WVRfLXJ6ZWZnWVE/view?usp=sharing Picture of cells incubated in tumeric extract]

[https://drive.google.com/file/d/0B1jaPHOkH_42aUJydUdoWVZOOVE/view?usp=sharing Methylene Blue control]

File:MeBlueControl 170417.jpg

2017-05-19T12:27:30Z

Bastien: Positive control of cell staining with Methylene Blue, Tumeric experiment, 17.04.17

Positive control of cell staining with Methylene Blue,

Tumeric experiment, 17.04.17

Tumeric

2017-05-19T12:24:17Z

Bastien:

Experiment 7avr17 

Aim:
To get a cell staining protocol that uses non-toxic (kitchen) ingredients 

To see if tumeric might stain cells.

1) dry down some cheek cells on a slide, to fix with EtOH

2) make solution of Tumeric in ethanol (doesn't seem to dissolve in saline) 

3) put a drop of tumeric solution on one patch, methylene blue on another and compare 

Is any color visible? 
What about after treatments for comet tests?

Anna and Bastien already counted cheek cells, and are making pads before RA joined in.
Had plan for an experiment with the osmotic shock and the detergent treatment already.
will try staining one of each with MeBlue and one with tumeric?

The tumeric did not seem to do much, if anything...

=== Galery ===

[https://drive.google.com/file/d/0B1jaPHOkH_42WVRfLXJ6ZWZnWVE/view?usp=sharing Picture of cells incubated in tumeric extract]

[https://drive.google.com/file/d/0B1jaPHOkH_42aUJydUdoWVZOOVE/view?usp=sharing Methylene Blue control]

AGiR! for genomic integrity

2017-05-19T12:19:28Z

Bastien:

[[File:MinilogoB.jpg |200px|thumb|]]
Genomic integrity is a new 'big picture' concept to aid public health efforts and basically includes all the molecular genetic details of cells!
So many things we commonly choose to do can impact genomic integrity, also potentially affecting future generations! 

At the Hackuarium, AGiR! hopes to really get some citizen science action going - developing DIYbio methods to readily assay *your very own* cells for DNA damage, using classic (circa 1980s) techniques. If these can still be sold in 'kits,' maybe it will also be simple to make them available to all for open source efforts?
 
Here is the basic plan: using inner cheek cells for micronuclei and comet assays!
 
Watch this space for more info and visit
[http://www.genomicintegrity.org the AGiR! site]

== Introduction ==
=== Context ===
This is a part of a Hackuarium project by AGiR! on genomic integrity, to find out more and help raise awareness in the public interest. 
This wiki page is under construction, and will help keep track as we develop open source simple protocols of two classic methods for quantitation of DNA damage in human cells, the micronuclei and comet assays.

=== People ===
'''Contact and lead:'''
*Rachel Aronoff - Founder and CSO of AGiR! Action for Genomic integrity through Research!

'''Participants:'''
*maybe you?!

=== Main Goals ===

Genomic integrity is a big picture concept which includes basically all the molecular genetic details of cells (from RNAs to DNAs and the epigenetic marks for proper gene expression), and thus is a topic that can be explored in several ways. For the genomic integrity project at the Hackuarium the focus will be on directly quantifying DNA damage. Comet and micronuclei assays were developed in the 1980s and publications about use of these assays on human cheek cells are already in the literature. These methods, as open source protocols, will encourage citizen science and raise awareness. Additionally, they can provide an easily "workshopable" way to show people more about their own cells, while learning how to assess DNA damage. In other words, this experiment can be used as a gateway, to demonstrate and raise awareness for everyone on the damage possible from many common substances or activities - on '''your own cells, your own genetic information'''! In the scope of Hackuarium activities, mammalian cell cultures are still (april 2016) a no go, but freshly isolated cheek cells from individuals can be used to do tests on DNA damaging compounds or activities. Such 'ex situ' assays might seem more attractive, especially if potentially toxic compounds are tested! (but the caveat is that it is only after the metabolism of some compounds that the DNA-damaging effects are 'revealed' - for example alcohol to DNA-damaging aldehydes…) For this reason, 'citizen scientists' will be encouraged to first get baseline measures on their cells, then do a 'trial' and test its effects (i.e. daily swimming, chocolate or blueberry consumption, or some other activity). The comet and micronuclei assay results can also be compared after a 'wash-out' stage, without the given trial activity… These would all provide 'biomonitoring' results about the given 'intervention' from baseline conditions. Another possibility with these assays are to perform ''ex situ'' experiments on your own cells, as mentioned, which would be giving acute genotoxicity information... In order to further investigate compounds that might cause DNA damage, a classic test of bacterial reversion (i.e. the Ames test) or on bulk DNA may also be nice to establish at the Hackuarium. If enough people follow the 'open source' protocol(s) that we develop to follow controlled exposure protocols or test substances directly on their isolated cheek cells, and clearly significant effects are observed, we may even be able to corroborate or refute previous works in the literature. (Please go to the bottom of the page, for one concrete example being considered.) Your ideas and comments are welcome throughout btw!

=== Objectives ===

* Obtain ready results with cheek cells for the comet and micronuclei assays

* Put together the open source protocols for each method, with suggestions for standard tests and controls

* Demonstrate methods and 'share the love' with other biohacker groups (La Paillasse etc.)

* Study the feasibility of further methods that could contribute to this project - i.e. an assay on either bacterial or bulk DNA

'''Cheek cells are continually renewing epithelial cells and are about 50 micrometers in diameter.''' [[File:Cells3.jpg|200px|thumb|right|Three cheek cells imaged on the scope at the Hackuarium. Some methylene blue stain may be visible in the most 'in focus' nucleus - left, middle cell. ]]

== Background & Inspiration ==
Here is the link to the Prezi Rachel presented (13apr16) at an Open hackuarium night!
[http://prezi.com/r9zepj_-emx-/diybio-for-genomic-integrity/ Prezi on DIYbio for Genomic integrity!]

=== Some theory ===
More on comet cells and micronuclei to come soon!
But in the meantime, here are the references to the cheek cell papers mentioned in the presentation (and in the prezi): 
Szeto et al (2005) Mutation Research 578:371-381 
(In this paper, green tea is also shown to be protective.) 
dos Santos Rocha et al (2014) Genetics and Molecular Biology, 37(4)702-707 
(This paper looks at micronuclei in cheek cells (and also analyses apoptosis) from mouthwash and alcohol users.) 

''To note: the two assays proposed would be analysing only limited aspects of 'genomic integrity' at the DNA level - large scale chromosomal disruption in the micronuclei assay and double-stranded breaks with the comet assay! There are many more methods that could look at other aspects (particular nucleotide lesions, reactive oxygen species and inflammation, gene expression, microRNAs, etc.) of genomic integrity!''

=== Inspiration / Similar projects ===
Inspiration:
* [https://exogenbio.com/] Sylvain Costes and Jon Tang made this great effort in 2014, based upon sub-cellular fluorescence quantification of DNA Damage Response components, but their blood draw kit was a bit tricky for some, and Sylvain told me the results were not so reliable after these fixed cells arrived in the mail, as opposed to their results when they had them 'fresh.'
* [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246922/] This is a Frontiers in Genetics article with a nice focus on using inner cheek cells and other epithelial cells for DNA damage assessment, also from 2014.
* [http://www.sciencedirect.com/science/article/pii/S138357181300003X This paper] describes results of DNA damage scoring in soy bean farm workers exposed to various pesticides, with DNA damage scored via comet assays of blood cells and micronuclei assays of cheek cells.

=== Setup description ===
General principle for both assays: looking at treated cells on slides through a microscope, imaging and quantitating the effects (i.e. counting number of cells with micronuclei relative to the total number of cells, or determining the length of the 'comet tail' - the longer the 'tail' the more DNA breaks - on average). 
The comet assay requires more treatments and a short 'electrophoresis' step, as compared to the micronuclei assay. 
For the purpose of cheek cell comet assays, use of a special chamber with mini-electrophoresis capacity, capable of microscopic investigation, is being planned.

=== Necessary Materials ===
*Interested citizen scientists
*A toothbrush for gentle collection of the cheek cells
*Lab equipment for the cheek cell 'comet' and micronuclei assays
::* Microscope, glass slides
::* Lab 'consumables' pipets and tips, beakers
::* Agarose, and gel running apparatus for embedding cells, treatments (triton, protease'*, EDTA'**, buffer solutions, etc.) and nucleoid migration (for the comet assay)
:::* Mercury Lamp Setup and filters for fluorescent dye detection.
:::* Could also try to design a transilluminator suitable for cellular resolution (UV/Fluo as usual for comets) when imaging (to look for open source models)

::* Other reagents and histochemical stains (methylene blue)
::* Hydrogen peroxide for positive control cells (oxidative damage)
::* Antioxidant molecules as possible 'protective' intervention . (i.e. green tea as in Szeto et al...)

'*Proteinase K is recommended in buccal cell protocols, but DIY alternatives are being considered, like tablets for contact lens cleaning 
'** To protect the DNA from digestion

** More details to come (What, size | quantity | where to get it | price / unit)
Very likely, working in pairs will be ideal for workshops based on these assays, since 'self-reporting' is generally so biased!
(AGiR! is actively investigating this issue, too, since it has effects on both sides of the equation, for producers as well as consumers!)

==== Parameters for the Open Source Protocol (still under development!) ====
;To get simplest protocol established, compared with the standard lab techniques

then for each individual:
obtain baseline values (number of micronuclei per thousand cells, or average comet tail lengths/category distributions)
Ideally to get at least 3-5 independent measurements first
Then, for 'in situ' experiments to vary some lifestyle component, and repeat the assays again. (i.e. exercise, diet, exposures...)
Compare the obtained values - baseline vs. intervention...

; To measure/ observe
: p1 | number of micronuclei per thousand cells
: p2 | categories of comets (0-5...)

; Constants (Key elements of set-up)
: c1 |
: c2

;To get Open Source protocol out to the DIT Research communities

Then get workshops running, to familiarize people to the technique, and start gathering acquired baselines for individuals, who will either do ex situ experiments on their own cells, or vary something in their lifestyle (e.g. eat chocolate or blueberries, go running, stop drinking, etc.), and then start collecting 'big data' as more people join in this effort!

=== Protocol (Plan) ===
to do both comet cell and micronuclei assays by the simplest possible methods, with the bare minimal laboratory reagents/infrastructure...

==== Startup ====
To get going with classic methods, on cheek cells, esp following Szeto et al protocol comparisons for the comet assays, and staining fixed cells for the micronuclei...

==== Phase 1 ====
Try proteinase K only treatments (no trypsin). Cells simply dissolved away first few times, never seemed feasible to even run the electrophoresis step!
 
On the 4th set of attempts (2dec16), by watching carefully as they were being digested, the reaction could be stopped early enough for some visible nucleosomes to remain. 
Simply running the slides for the electrophoresis step in TBE, allowed the formation of the first cheek cell 'comets' in the Hackuarium! (thanks to Niko at DMF for letting me go on the departmental scope very quickly that afternoon! :)
 
[[File:CheekCellComet 2dec16slide2x10GFPb.jpg]] This cheek cell 'comet' was imaged by epifluorescence after staining with SYBR-Safe dye. 
[[File:ControlCheekCells_sybrsafe_2dec16slide1x10bGFP.jpg]] These are control cheek cells that were embedded and treated with all buffers, but never exposed to proteinase K and not run during the electrophoresis step. 
[[File:H2O2treatedCheekCell 2dec16positivecontrol.jpg]] This 'positive control comet' was obtained after treatment of embedded cheek cells also with hydrogen peroxide, before the proteinase K step. 
More to come! Who else wants to try?

=== Lab Notebook & News (Real) ===
Protocols and measurements to be linked from here: [http://wiki.hackuarium.ch/w/AGiR!_for_genomic_integrity/cometassaydev comet protocol development page]

== Run / prototype #2==

What's new compared to #1: getting the comets to show up with a non-fluorescent staining protocol (if possible)!

== Run / prototype #3==
What's new compared to #2:
Development of a microfluidic chip in a slide with miniaturised electrode links, to run the comet assays in a miniformat, that can then be directly examined on a basic microscope...
Design and implementation of this cheek cell comet device may be a key element for a crowd funding campaign (aiming for September 2017).

== Results / Feedback from use ==
== Really fun initial citizen science trial - testing chocolate's effects on genomic integrity ==

As one fun standard protocol for the open source 'citizen science' procedure, which might help resolve controversies in the literature even - for instance, about flavanols in chocolate and what else they might be good for (cardiovascular health, cognition, etc…) - AGiR! is considering proposing to test high and low flavanol chocolate's effects on genomic integrity! This will form one part of the initial citizen science trials for this project! ''Other potential plans include testing cells in the context of performing activities thought of as 'good' or 'bad' for you, and also testing the cells in isolation (ex situ assays)...'' In terms of chocolate: There have been two publications on limited numbers of subjects in terms of flavanols from chocolate protecting skin cells from UV light, based on a sort of 'quantitative skin burn' method - measuring the so-called minimal erythema dose).
Here are the abstracts from two interesting publications in that regard. http://www.ncbi.nlm.nih.gov/pubmed/19735513 http://www.ncbi.nlm.nih.gov/pubmed/24970388
 One of course found a significant effect and the other no significant UV protection of skin. (However a 'Net temple elasticity increased slightly but significantly by 0.09 ± 0.12 mm in the HFC group at 12 weeks compared to 0.02 ± 0.12 mm in the LFC group (MD: 0.06; 95% CI: 0.01 to 0.12 )' and this could be very interesting for many of us! 30g of chocolate a day was the dose, for 12 weeks and a 3 week washout in this study. [http://nutritionj.biomedcentral.com/articles/10.1186/1475-2891-13-66 Have a look]! - They even discuss the several reasons that the results are not strictly comparable between the two studies. Trial design is so crucial! )

Maybe in the terms of this genomic integrity project for the Hackuarium, using cheek cells to assess both basal damage levels and damage levels in response to tests of ''ex situ'' effects of differing concentrations of hydrogen peroxide, for instance, after eating chocolate with high or low flavanol levels, will be more meaningful than that 'skin burn' model, based on just 30 people. The comet and micronuclei assays are extremely established and clearly quantitative. Should be fun! Furthermore, with citizen scientists everywhere involved, we may be able to get much bigger numbers than for those two previous works. 
In the meantime… 
A collaboration with a very keen 'chocolatier' from Gruyere may help us!
We will try to see which of his dark chocolates contains the most flavanols (and perhaps he will modify some steps in the production process to increase the levels found!) and had a [https://www.facebook.com/photo.php?fbid=10210066414961608&set=o.543862129060777&type=3 'degustation' of his grand cru chocolates yesterday! (25 may 2016)] 

If you are interested in more information and a closer look at the results (in a nutshell, no significant preference was found for the four different chocolates provided, but there was a slight trend leading to two of the four seeming to be more preferred! Additionally, eating any of these magnificent chocolates made a commercial brand less attractive for many participants!), here is a page with further info about this: [[chocolate tasting 2016]]

As we are located in the land of chocolate - Switzerland - what could be better? Hoping people will like the idea! For us all!

== Recommendation + next steps ==
Check out the [http://wiki.hackuarium.ch/w/Moss_Menageries_with_AGiR! Moss Menagerie] project also,
and
spread the word!
www.genomicintegrity.org

[[Category:Work In Progress]]
[[Category:Manual]]

AGiR! for genomic integrity/cometassaydev

2017-05-19T12:15:24Z

Bastien: /* Experiments */

This is the page for concrete aspects of the cheek cell comet assay protocol...

[[File:CheekCellComet 2dec16slide2x10GFPb.jpg]]

==Experiments==
* Cheek Cell comets
* Test of [[Tumeric | Tumeric for staining]] on cheek cells (7avr17)
* Test of [[SDSorDishSoapforPK | SDS vs Dish soap vs Standard]] protocol

==Conditions for Comet Cell Assays ==

*tests of pbs vs saline
*tests for optimal numbers of cells
*tests of filtration vs gravity for eliminating big clumps of cells (1st attempt with lab 40micron filter run 8march2017, with Anna and Bastien)
*tests for staining dyes
*control conditions - negative control with no protease treatment, positive control with H2O2
*tests of non-lab 'kitchen sink' components for reactions (dish soap, contact lens cleaner, etc...)

==Current 'best' protocol==

1) isolate cells in saline solution (0.9% NaCl in water) after gentle toothbrush collection from inner cheek (rinse mouth with fresh water before 'harvest') 
- about 10ml solution 
Let biggest clumps settle away, and take remaining solution to pellet most cells
(2ml eppi tube, 2k rpm, 2min), repeating until all solution is pelleted. 
Resuspend pellets in about 200 microliters of fresh saline and estimate cell concentration and quality under scope (can use hemocytometer for counting) 

3) embed cells in 1% low melting point agarose (made up in standard saline solution)
aiming for about 20,000 cells to be added to 100 microliters molten agarose (kept at 42oC) 
mix and put on clean microscope slide using a clean coverslip to flatten it in place. (can flatten the cell suspension on top of a pre-made agarose base, if desired, see below) 

4) treat cell patches with first solutions: saline only, for test cases (3-5 replicates ideally); and H2O2 (0.01%) for positive control case. 

5) equilibrate all cell patches with Proteinase K (PK) buffer (for 2ml: 40 microliters 0.5M Tris pH8, 400 microliters 0.5M EDTA, 1ml 5M NaCl, 20 microliters 1% Triton, 0.54ml H2O) 
x2 changes (drops of liquid, about 20-50 microliters depending on patch sizes) 

6) treat cells with PK+ solution (final PK concentration at 0.5mg/ml: 5 microliters of 20mg/ml PK stock in 200 microliters PK buffer) 
''keep at least one cell patch without this treatment as your negative control!'' 
RT incubation (watch under scope to see cells 'disappearing' leaving nucleoids - usually only about 20 minutes max) 

7) as treatment seems sufficient, apply 1X TBE pH9 (alkaline treatment) to cell patches 
2x 5 min 

8) equilibrate all patches with normal 1X TBE 
3x 2 min 

9) run the electrophoresis step: 12V 15 min (only 2 slides will fit easily in the gel box currently used in the lab, but other possibilities exist) 

10) staining and imaging (most success with SYBR safe dye, diluted 1/10,000 in TBE for staining) and epifluor imaging ('green' channel) 

==Ideas for protocol improvement==

Hoping to assess alternatives that would form a nicely colored DNA 'comet tail' for easier DIY image acquisition/analyses, but neither Hemalum after a MeOH:acetic acid (3:1) fix nor methylene blue aqueous seem very useful at all in this regard... More testing and ideas are very welcome! 
 
In the meantime, DIYmicroscopes that can use a blue LED excitation light should be worked upon too!! 
 
If you have trouble removing the coverslip from the patch of embedded cells without messing it up, you can add buffer first, or lose patches during manipulations, you can make an initial patch on the slide before adding the cell suspension in agarose. 
Use two slides covered with tape and 2 more slides, as in [http://www.wormbook.org/chapters/www_intromethodscellbiology/cellfig1.jpg Monica Driscoll's old WormBook method]. (of course, instead of worms in solution, put your embedded cheek cells in molten agarose on top of pre-made patch, then flatten with coverslip as above. 
 
One patch can be cut into sections, for quick tests in different conditions, for example: middle untreated, one side for test, other side for positive control. 
 

== Ideas for 'Comet Chip' development as an 'automated' system ==
an open source microfluidic device is the plan! (with built-in mini-electrodes and microcontroller links)

More to come soon!!
 
Back to the [http://wiki.hackuarium.ch/w/AGiR!_for_genomic_integrity Genomic integrity project page at the Hackuarium]
 
To the [http://www.genomicintegrity.org/ AGiR! site]

AGiR! for genomic integrity/cometassaydev

2017-05-19T12:13:15Z

Bastien: /* Experiments */

This is the page for concrete aspects of the cheek cell comet assay protocol...

[[File:CheekCellComet 2dec16slide2x10GFPb.jpg]]

==Experiments==
* Cheek Cell comets
* Test of [[Tumeric | Tumeric for staining]] (7avr17)
* Test of [http://wiki.hackuarium.ch/w/Tumeric Tumeric] on cheek cells
* Test of [http://wiki.hackuarium.ch/w/SDSorDishSoapforPK SDS vs dish soap vs standard] protocol

==Conditions for Comet Cell Assays ==

*tests of pbs vs saline
*tests for optimal numbers of cells
*tests of filtration vs gravity for eliminating big clumps of cells (1st attempt with lab 40micron filter run 8march2017, with Anna and Bastien)
*tests for staining dyes
*control conditions - negative control with no protease treatment, positive control with H2O2
*tests of non-lab 'kitchen sink' components for reactions (dish soap, contact lens cleaner, etc...)

==Current 'best' protocol==

1) isolate cells in saline solution (0.9% NaCl in water) after gentle toothbrush collection from inner cheek (rinse mouth with fresh water before 'harvest') 
- about 10ml solution 
Let biggest clumps settle away, and take remaining solution to pellet most cells
(2ml eppi tube, 2k rpm, 2min), repeating until all solution is pelleted. 
Resuspend pellets in about 200 microliters of fresh saline and estimate cell concentration and quality under scope (can use hemocytometer for counting) 

3) embed cells in 1% low melting point agarose (made up in standard saline solution)
aiming for about 20,000 cells to be added to 100 microliters molten agarose (kept at 42oC) 
mix and put on clean microscope slide using a clean coverslip to flatten it in place. (can flatten the cell suspension on top of a pre-made agarose base, if desired, see below) 

4) treat cell patches with first solutions: saline only, for test cases (3-5 replicates ideally); and H2O2 (0.01%) for positive control case. 

5) equilibrate all cell patches with Proteinase K (PK) buffer (for 2ml: 40 microliters 0.5M Tris pH8, 400 microliters 0.5M EDTA, 1ml 5M NaCl, 20 microliters 1% Triton, 0.54ml H2O) 
x2 changes (drops of liquid, about 20-50 microliters depending on patch sizes) 

6) treat cells with PK+ solution (final PK concentration at 0.5mg/ml: 5 microliters of 20mg/ml PK stock in 200 microliters PK buffer) 
''keep at least one cell patch without this treatment as your negative control!'' 
RT incubation (watch under scope to see cells 'disappearing' leaving nucleoids - usually only about 20 minutes max) 

7) as treatment seems sufficient, apply 1X TBE pH9 (alkaline treatment) to cell patches 
2x 5 min 

8) equilibrate all patches with normal 1X TBE 
3x 2 min 

9) run the electrophoresis step: 12V 15 min (only 2 slides will fit easily in the gel box currently used in the lab, but other possibilities exist) 

10) staining and imaging (most success with SYBR safe dye, diluted 1/10,000 in TBE for staining) and epifluor imaging ('green' channel) 

==Ideas for protocol improvement==

Hoping to assess alternatives that would form a nicely colored DNA 'comet tail' for easier DIY image acquisition/analyses, but neither Hemalum after a MeOH:acetic acid (3:1) fix nor methylene blue aqueous seem very useful at all in this regard... More testing and ideas are very welcome! 
 
In the meantime, DIYmicroscopes that can use a blue LED excitation light should be worked upon too!! 
 
If you have trouble removing the coverslip from the patch of embedded cells without messing it up, you can add buffer first, or lose patches during manipulations, you can make an initial patch on the slide before adding the cell suspension in agarose. 
Use two slides covered with tape and 2 more slides, as in [http://www.wormbook.org/chapters/www_intromethodscellbiology/cellfig1.jpg Monica Driscoll's old WormBook method]. (of course, instead of worms in solution, put your embedded cheek cells in molten agarose on top of pre-made patch, then flatten with coverslip as above. 
 
One patch can be cut into sections, for quick tests in different conditions, for example: middle untreated, one side for test, other side for positive control. 
 

== Ideas for 'Comet Chip' development as an 'automated' system ==
an open source microfluidic device is the plan! (with built-in mini-electrodes and microcontroller links)

More to come soon!!
 
Back to the [http://wiki.hackuarium.ch/w/AGiR!_for_genomic_integrity Genomic integrity project page at the Hackuarium]
 
To the [http://www.genomicintegrity.org/ AGiR! site]

User:Bastien

2017-05-19T11:36:23Z

Bastien:

== Who I am ==

I am an EPFL Master student majoring in Bioengineering. Lately, i've been interested in microfabrication, particularly of BioMEMS and point-of-care diagnostics devices.

== Why I love Hackuarium ==

Hackuarium is an opportunity to work with amazing and open-minded people, on projects that show a variety unobtained on a regular campus-lab.

== How to contact me ==

Email me anytime

== What I am currently working on ==

My main project is to help the [[AGiR! for genomic integrity/cometassaydev | genomic integrity project]].

== Test of wiki formatting I am conducting ==

=== Subtest ===

[[Main Page | Internal Link]]

User:Bastien

2017-05-19T11:34:40Z

Bastien: /* Subtest */

== Who I am ==

I am an EPFL Master student majoring in Bioengineering. Lately, i've been interested in microfabrication, particularly of BioMEMS and point-of-care diagnostics devices.

== Why I love Hackuarium ==

Hackuarium is an opportunity to work with amazing and open-minded people, on projects that show a variety unobtained on a regular campus-lab.

== How to contact me ==

Email me anytime

== What I am currently working on ==

My main project is to help the [http://wiki.hackuarium.ch/w/AGiR!_for_genomic_integrity genomic integrity project].

== Test of wiki formatting ==

=== Subtest ===

[[Main Page | Internal Link]]

[[AGiR! for genomic integrity/cometassaydev | genomic integrity]]

User:Bastien

2017-05-19T11:32:47Z

Bastien: /* Subtest */

== Who I am ==

I am an EPFL Master student majoring in Bioengineering. Lately, i've been interested in microfabrication, particularly of BioMEMS and point-of-care diagnostics devices.

== Why I love Hackuarium ==

Hackuarium is an opportunity to work with amazing and open-minded people, on projects that show a variety unobtained on a regular campus-lab.

== How to contact me ==

Email me anytime

== What I am currently working on ==

My main project is to help the [http://wiki.hackuarium.ch/w/AGiR!_for_genomic_integrity genomic integrity project].

== Test of wiki formatting ==

=== Subtest ===

[[Internal Link | Main Page]]

User:Bastien

2017-05-19T11:32:12Z

Bastien: /* Test of wiki formatting */

== Who I am ==

I am an EPFL Master student majoring in Bioengineering. Lately, i've been interested in microfabrication, particularly of BioMEMS and point-of-care diagnostics devices.

== Why I love Hackuarium ==

Hackuarium is an opportunity to work with amazing and open-minded people, on projects that show a variety unobtained on a regular campus-lab.

== How to contact me ==

Email me anytime

== What I am currently working on ==

My main project is to help the [http://wiki.hackuarium.ch/w/AGiR!_for_genomic_integrity genomic integrity project].

== Test of wiki formatting ==

=== Subtest ===

[Interanl Link | Main Page]

User:Bastien

2017-05-19T11:30:49Z

Bastien:

== Who I am ==

I am an EPFL Master student majoring in Bioengineering. Lately, i've been interested in microfabrication, particularly of BioMEMS and point-of-care diagnostics devices.

== Why I love Hackuarium ==

Hackuarium is an opportunity to work with amazing and open-minded people, on projects that show a variety unobtained on a regular campus-lab.

== How to contact me ==

Email me anytime

== What I am currently working on ==

My main project is to help the [http://wiki.hackuarium.ch/w/AGiR!_for_genomic_integrity genomic integrity project].

== Test of wiki formatting ==

=== Subtest ===

User:Bastien

2017-05-19T11:04:53Z

Bastien:

== Who I am ==

I am an EPFL Master student majoring in Bioengineering. Lately, i've been interested in microfabrication, particularly of BioMEMS and point-of-care diagnostics devices.

== Why I love Hackuarium ==

Hackuarium is an opportunity to work with amazing and open-minded people, on projects that show a variety unobtained on a regular campus-lab.

== How to contact me ==

Email me anytime

== What I am currently working on ==

My main project is to help the [http://wiki.hackuarium.ch/w/AGiR!_for_genomic_integrity genomic integrity project].