Michael Raines Big dumb monkey as Big dumb monkey …. Becka Adams Self as Self. Reno Ministrelli Self as Self. Sian Proctor Self as Self. George Willis Self as Self. Thom Beers Narrator as Narrator. Andre Johnson Actor as Actor …. Ali Olomi Trader as Trader.
Damien Puckler Marauder 2 as Marauder 2. Jerry Trimble Marauder as Marauder. Lorin McCraley Marauder as Marauder. Mushari Biker as Biker …. Elizabeth Anweis Survivor as Survivor. Farshad Farahat Trader as Trader. Wayne Douglas Morgan Abductor as Abductor. More like this. Watch options. Storyline Edit. Add content advisory.
Did you know Edit. User reviews 13 Review. Top review. Fact or Fiction. OK, so I'm just going to make this short: The premise is that it's essentially a live action role play scenario. Yes, some of their successes are a bit far fetched, but to be real, that doesn't make for an entirely compelling narrative. We're not talking about Survivor, we're not talking about realism here. What we're experiencing is a social experiment of a variety. The first season was more believably populated by people who I would trust in a survival situation, definitely.
Would a ragtag group be able to pull it together enough to make it through day-to-day? Would they be able to use enough common sense and basic knowledge to complete complicated and confusing tasks? Could they, ultimately, rely on one another for survival? Would it be likely or even realistic for that to be the case: Of course not.
As for staged scenarios, I really can't argue much on that, except to say it makes sense to have tasks be staged as well as to have the raiders and non-colony members be actors and have their reactions staged. A colony is considered to be 50 cells or more and they are only visible under a microscope. As this assay is typically carried out over a long period of time, you will need to change your cell media. Cells will be at a very low confluence to start with, so you will not need to change the media as regularly as normal.
However, if you are seeding and then treating cells with a drug or compound, it is important to consider its half-life and add fresh treatment as needed. It is often the colonies under the control conditions that grow the fastest and therefore you can use these cells as an indication of your experimental endpoint, i.
It is important to end the experiment for all treatment conditions at the same time. Once you have reached your experimental endpoint, cells need to be washed gently with PBS add the PBS to the side of the well to not disrupt the colonies , fixed, and stained with the DNA intercalating dye, crystal violet 0. Removing the excess stain can be messy. The best technique is to gently dunk the plates in beakers immersed with water until all excess stain has been removed and you are only left with bright-purple colonies.
Stained colonies can be counted up to 50 weeks after staining. Take high-resolution pictures of your wells to use for analysis, presentations, and publication figures.
In the incubator, time-lapse imaging can provide kinetic information rather than an endpoint, which can provide more detailed information about when the treatment starts to affect the cells. Label-free image analysis can be used to detect colonies , evaluate their size and circularity, as well as pinpoint when colonies are starting to merge and optimize the assay accordingly. It really is as simple as manually counting your colonies for each treatment condition and representing the data as a survival curve you should use at least three biological repeats for your curve.
A survival curve requires the surviving fraction SF of treated cells this includes a ratio of colonies formed to cells seeded to be calculated and plotted against the treatment dose. Before you can calculate the SF, the plating efficiency PE of your cells needs to be determined as different cell lines have different plating efficiencies and this affects the survival fraction calculation. PE is the ratio of the number of colonies to the number of cells seeded in your untreated cells 1.
Figure 1 shows the PE and SF formulae and an example of a survival curve. The manual counting of colonies is tiresome and can be prone to bias, so a number of freely available computerized image analysis tools have been developed for analyzing clonogenic assay images quickly and objectively. A worthy mention is the freely available software package developed by Brzozowska et al.
An alternative to counting and quantifying individual colonies is determining the percentage of the well area that is covered by colonies colony area percentage to quantify clonogenic cell growth.
This is a useful tool to use if you have merged colonies that are difficult to count by eye or by colony counting software or if you want a quick and high-throughput analysis method. Figure 2 Freely available clonogenic assay measurement tools. A Brzozowska et al. A recent paper by Mayr et al.
This method enables comprehensive experimental setups using a well plate, it is label-free, has no staining endpoint, and the analysis is semi-automatic Figure 3 8. Furthermore, time-resolved, non-endpoint confluence measurement of the same well showed that semi-automatic analysis was suitable for determining colony number and mean size.
Figure 3 Mayr et al. The graph shows quantification of clonogenic growth at different time points and the images show specific wells with confluence detection. Green spots represent areas detected by a confluence reader and orange arrows indicate merged colonies. This clonogenic assay method provides a time- and cost-effective alternative to the standard clonogenic assay protocol. With no endpoint fixation and staining required, this protocol enables continuous monitoring and analysis of clonogenic growth.
Furthermore, additional metrics about the kinetics of colony growth can also be extracted during the experiment. Ultimately, Mayr et al. A label-free, non-endpoint, and automated solution for your clonogenic assays is the CytoSMART Omni with its colony detection algorithm. Colony formation is measured over time across an entire well with colony count, size, and circularity readouts. Your cells do not have to leave the incubator and cellular kinetic information during the process of colony formation can be obtained Figure 4.
The image shows a full well after the colony detection algorithm was run with clusters of cells highlighted by the orange colony mask. Colony count, size, and circularity were measured throughout the duration of the assay and are shown in the graphs.
A clonogenic assay, also known as a colony formation assay is an in vitro cell survival assay. This blog highlights: Clonogenic assay applications How to perform a clonogenic assay with important considerations to make along the way Quantifying your colonies and plotting a survival curve Using label-free microscopy and confluence detection to assess colony count and size using automated image quantification The clonogenic survival assay Clonogenic assays are widely used in the field of cancer research as the formation of clones is interpreted as a trait of cancer cells with tumor-initiating capabilities.
How to perform a clonogenic assay The information in this section is adapted from Franken et al. Essentially, there are two different ways to perform a clonogenic assay: 1 Cells can be seeded at low densities and then treated to examine the effect of the treatment on the clonogenicity of cells.
Traditional protocol for clonogenic assays Figure 1 A summary of a traditional clonogenic protocol where cells are seeded, treated, and then clonogenicity assessed.
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