User:Penelope Sky/sandbox

11/21 Thanks!! Added contribution to main article. 11/17

Beautiful work!!! -

11/13 Response to Shannon's comments:

Cell sorting is the process of taking cells from an organism and separating them by desired groupings based on morphology and desired research. The cells are labelled and tagged to identify the areas of interest and their affect. The most commonly used methods are, FACS (fluorescent activated cell sorting) and MACS (magnetic activated cell sorting). These methods are mostly used in hematology, cytogenetics and stem cell research laboratories.

The FACS method utilizes fluorescent dyes and suspends the desired cells in droplets. Diagram A shows FACS of negative cell selection (undesired group) and diagram B shows FACS of positive cell selection (desired group). The MACS method uses magnetic beads called microbeads that are paired with a group of cells, then either placed within a magnet or exposed to a magnetic field after being incubated or shaken in a buffering solution. The cells paired with microbeads attach themselves to the magnetic field and the non-paired cells are removed. This process can then be repeated to continue removal of the selected cells.

The MACS method has been used in assistance with reproduction (artificial insemination), retinal transplant treatment, and success with use of neural cell cultures. In the case of assisting reproduction, apoptotic sperm cells (dead or damaged cells) are separated out so more non-apoptotic sperm (non-fragmented) cells can be collected and used to increase the subject's chances of fertility. This type of treatment has shown to be more effective when done repeatedly, increasing the amount of non-apoptotic cells present during insemination.

A 2018 study done in France (with the support of multiple individuals and agencies including: the Insitut de la Vision in Paris and the Retina France Association) used rats and the MACS method to show that photoreceptors (cells in the retina which respond to light) may be transplanted to cure blindness. In this process, the microbeads were attached to the CD73 enzyme to assist in the separation of PRs (photoreceptors) from retinal organoids. When a CD73+ antigen expressed itself with RCVRN+ cells (calcium-binding proteins in the eye), it showed researchers that this combination of CD73+ and RCVRN+ could be used with post-mitotic PR precursors for repair. Although the study could not verify success in humans, they have the foundation for further research based on the success of pairing non-damaged photoreceptors with a CD73 antigen and the transplantation in rats. This success in cell separation and pairing through transplantation shows promise for a potential cure for retinal diseases including total blindness. So far, only partial vision repair has been reported.

MACS has also shown promise when used with neural stem cell cultures. These cultures are especially difficult to work with because live brain cells are sensitive and tend to contaminate each other. In order to get clearer results, labs need cleaner materials, meaning more pure neural progenitor cell (NPC) lines. A study done in 2019 (with the funding support of New York Stem Cell Foundation and the Association for Frontotemporal Degeneration) found MACS to be a cheap, simple way to yield such purity with minimal damage to the cell lines, therefore maintaining better quality cells, collecting more homogeneous NPCs, and increasing their chances of finding effective treatments for neurological disorders. They used both the MACS and FACS methods to filter out CD271- (useful markers for mesenchymal stem cells) and CD133+ (markers for cancer stem cells) to compare viability of each method.

References:

11/6 Peer review response:

11/10 Shannon's comments

Fan-freaking-tastic job!

Just a couple of questions:

What did the magnetic beads stick to in the retinal study? Why did they need to add the CD73 antigen to the cells before transplantation?

Also what are they filtering out of the clean NPC samples with the magnetic beads?

Great work!

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Cell sorting is the process of taking cells from an organism and separating them by desired groupings based on morphology and desired research. The cells are labelled and tagged to identify the areas of interest and their affect. The most commonly used methods are, FACS (fluorescent activated cell sorting) and MACS (magnetic activated cell sorting). These methods are mostly used in hematology, cytogenetics and stem cell research laboratories.

The FACS method utilizes fluorescent dyes and suspends the desired cells in droplets. Diagram A shows FACS of negative cell selection (undesired group) and diagram B shows FACS of positive cell selection (desired group). The MACS method uses magnetic beads called microbeads that are paired with a group of cells, then either placed within a magnet or exposed to a magnetic field after being incubated or shaken in a buffering solution. The cells paired with microbeads attach themselves to the magnetic field and the non-paired cells are removed. This process can then be repeated to continue removal of the selected cells.

The MACS method has been used in assistance with reproduction (artificial insemination), retinal transplant treatment, and success with use of neural cell cultures. In the case of assisting reproduction, apoptotic sperm cells (dead or damaged cells) are separated out so more non-apoptotic sperm (non-fragmented) cells can be collected and used to increase the subject's chances of fertility. This type of treatment has shown to be more effective when done repeatedly, increasing the amount of non-apoptotic cells present during insemination.

A 2018 study done in France (with the support of multiple individuals and agencies including: the Insitut de la Vision in Paris and the Retina France Association) used rats and the MACS method to show that photoreceptors (cells in the retina which respond to light) may be transplanted to cure blindness. Although the study could not verify success in humans, they have the foundation for further research based on the success of pairing non-damaged photoreceptors with a CD73 antigen and the transplantation in rats. This success in cell separation and pairing through transplantation shows promise for a potential cure for retinal diseases including total blindness. So far, only partial vision repair has been reported.

MACS has also shown promise when used with neural stem cell cultures. These cultures are especially difficult to work with because live brain cells are sensitive and tend to contaminate each other. In order to get clearer results, labs need cleaner materials, meaning more pure neural progenitor cell (NPC) lines. A study done in 2019 (with the funding support of New York Stem Cell Foundation and the Association for Frontotemporal Degeneration) found MACS to be a cheap, simple way to yield such purity with minimal damage to the cell lines, therefore maintaining better quality cells, collecting more homogeneous NPCs, and increasing their chances of finding effective treatments for neurological disorders.

References:

10/19 peer review by Kameryn Landry

Hi Pauline-

There are lots of interesting topics in your contribution, however there are a couple spots that confused me. I am unclear on the definition of what cytogenics is. The diagrams are very helpful in understanding what FACs and MACs methods are. One thing I think you could add to give more information would be where and when the study on rats was done. You stated that it was completed in 2018, but not much further detail. Also, did the study show that transplanting photoreceptors is able to cure complete blindness? Or just near-blindness such as myopia? Another definition that was unclear is on neural progenitor cell lines. Does this process contribute to treatment of neurological disorders?

Overall I think your contribution was very well written, without any spelling or grammatical errors. 10/20-10/21 Draft of contribution after reading instructions email: Cell sorting is the process of taking cells from an organism and separating them by desired groupings based on morphology and desired research. The cells are labelled and tagged to identify the areas of interest and their affect. The most commonly used methods are, FACS (fluorescent activated cell sorting) and MACS (magnetic activated cell sorting). These methods are mostly used in hematology, cytogenetics and stem cell research laboratories.

The FACS method utilizes fluorescent dyes and suspends the desired cells in droplets. Diagram A shows FACS of negative cell selection (undesired group) and diagram B shows FACS of positive cell selection (desired group). The MACS method uses magnetic beads called microbeads that are paired with a group of cells, then either placed within a magnet or exposed to a magnetic field after being incubated or shaken in a buffering solution. The cells paired with microbeads attach themselves to the magnetic field and the non-paired cells are removed. This process can then be repeated to continue removal of the selected cells.

The MACS method has been used in assistance with reproduction (artificial insemination), retinal transplant treatment, and success with use of neural cell cultures. In the case of assisting reproduction, apoptotic sperm cells (dead or damaged cells) are separated out so more non-apoptotic sperm (non-fragmented) cells can be collected and used to increase the subject's chances of fertility. This type of treatment has shown to be more effective when done repeatedly, increasing the amount of non-apoptotic cells present during insemination.

A study done in 2018 on rats used the MACS method to show that photoreceptors (cells in the retina which respond to light) may be transplanted to cure blindness. Although the study could not verify success in humans, they have the foundation for further research based on the success of pairing non-damaged photoreceptors with a CD73 antigen and the transplantation in rats.

MACS has also shown promise when used with neural stem cell cultures. These cultures are especially difficult to work with because live brain cells are sensitive and tend to contaminate each other. In order to get clearer results labs need cleaner materials, meaning more pure neural progenitor cell lines. This study found MACS to be a cheap, simple way to yield such purity with minimal damage to the cell lines, therefore maintaining better quality cells for further treatment of neurological disorders.

References:

10/20 Another source for use of MACS: Bansal, R., Hinduja, R., & Desai, N. (2018). DNA Fragmentation Index and Magnetic Activated Cell Sorting: A Practical Approach. Practical Guide in Andrology and Embryology, 97. 10/20 I haven't been able to find anything to support the statement, "there are several types of magnetic cell sorting." The article does explain column based separation and column-free separation, but as far as I have read they all use a similar process.

As part of my contribution, I will remove the questionable sentence and replace with a description of the process. "Specific cells are paired with magnetic beads called microbeads, then either placed within a magnet or exposed to a magnetic field. The cells paired with microbeads attach themselves to the magnetic field and the non-paired cells are removed. This process can then be repeated to continue removal of selected cells." In some instances the cells are incubated with a buffering solution before being exposed to the magnetic field."

The removed cells can be for either negative or positive selection. Some examples of how they are used are: assistance in reproduction (artificial insemination) and transplants (replacement treatment)." 10/19 Completed training module: contributing images and media files. Found a couple images that could be useful in the cell sorting article.

10/9 Editing notes/ideas for Cell Sorting article contribution:

- Move diagram for MACS process to the MACS section of the article.

- Add more information to the MACS section to create better balance between MACS and FACS sections.

- Example of needed information for the MACS section: a citation and a list of the several types of magnetic cell sorting after the sentence "there are several types of magnetic cell sorting."

- Maybe add to both sections to include more detailed examples of how the two methods are used. Examples that the average reader could relate to or at least understand and have a better idea of how these methods are used and what for.

- Add more links to terms used or pieces of the processes that the average reader may not already know or be familiar with. For example, labelled or tagged cells. Could seem obvious, but a link to more information or a brief description would be helpful. There are other areas where I already feel like I understand this article much more because of being in this class. So identifying those areas where upon initially reading this article I was confused versus what I understand now, having gained more in depth knowledge of cells and their functions, will help identify areas where extra links could be added.

- Clarify the FACS diagram on dyes.

- Add to the buoyancy activated cell sorting (BACS) section. It's so small and insignificant looking that I forgot about it, which means the average reader probably would too. 10/2 Selected third reference for cell sorting article:

Reference notes for Cell Sorting article:

1.) Nitta, N., Sugimura, T., Isozaki, A., Mikami, H., Hiraki, K., Sakuma, S., ... & Fukuzawa, H. (2018). Intelligent image-activated cell sorting. Cell, 175(1), 266-276. Found on Google Scholar

2.) Bowles, K. R., T. C. W., J., Qian, L., Jadow, B. M., & Goate, A. M. (2019). Reduced variability of neural progenitor cells and improved purity of neuronal cultures using magnetic activated cell sorting. PLoS ONE, (3). Retrieved from http://search.ebscohost.com/login.aspx?direct=true&AuthType=shib&db=edsgov&AN=edsgcl.580304579&site=eds-live. Found in Puksta Library Search

3.) Gagliardi, G., Ben M’Barek, K., Chaffiol, A., Slembrouck-Brec, A., Conart, J.-B., Nanteau, C., … Goureau, O. (2018). Characterization and Transplantation of CD73-Positive Photoreceptors Isolated from Human iPSC-Derived Retinal Organoids. Stem Cell Reports, 11(3), 665–680. https://doi.org/10.1016/j.stemcr.2018.07.005 9/25 Added to MACS section of cell sorting article with a citation: "However, MACS has shown to be beneficial when used with NPC (neural progenitor cell) cultures in particular, as it is easier to manage and causes minimal damage to live cells.[8]" -Removed the citation link from this page for organizational purposes. It is the 2nd reference in my notes below and 8th reference in the article. 9/24-9/25 Reference notes for Cell Sorting article:

1.) Nitta, N., Sugimura, T., Isozaki, A., Mikami, H., Hiraki, K., Sakuma, S., ... & Fukuzawa, H. (2018). Intelligent image-activated cell sorting. Cell, 175(1), 266-276. Found on Google Scholar

2.) Bowles, K. R., T. C. W., J., Qian, L., Jadow, B. M., & Goate, A. M. (2019). Reduced variability of neural progenitor cells and improved purity of neuronal cultures using magnetic activated cell sorting. PLoS ONE, (3). Retrieved from http://search.ebscohost.com/login.aspx?direct=true&AuthType=shib&db=edsgov&AN=edsgcl.580304579&site=eds-live. Found in Puksta Library Search 9/24 - Completed training module: Adding Citations 9/12 - Evaluated article: Cell Sorting and Completed training module: Wikicode vs. Visual Editor 9/8 - Completed training module: Evaluating articles and sources. 9/3 - Completed training modules: Wiki Policies, Sandbox, Talk pages and Watchlists. 8/28 - I signed up for a wiki account. References: