I have a problem with number of cells in E0771 cell line . I usually use 100-2000 cells for sphere but I couldn't get good sphere.
The number of cells used for a sphere assay can vary depending on the specific cell line and experimental conditions. In the case of the E0771 cell line, the optimal number of cells for sphere formation may require some optimization. While there is no standard or fixed number of cells that universally guarantees successful sphere formation, a commonly used range for the E0771 cell line is 500-2000 cells per well.
However, if you are not obtaining satisfactory sphere formation with the range of 100-2000 cells, it is possible that other factors may be affecting the assay. Some factors to consider and troubleshoot include:
1. Cell density: Ensure that the cells are plated at the desired density in a suitable culture medium and appropriate culture conditions.
2. Culture conditions: The sphere-forming capability of cells can be influenced by various factors such as culture medium composition, presence of growth factors or supplements, and the presence of a suitable 3D culture system.
3. Passage number: The passage number of the cells can affect their behavior and sphere-forming ability. It is generally recommended to use cells at lower passages to maintain their characteristics.
4. Time and observation: Spheres may take some time to form, and they may not be visible or well-developed immediately after plating. Allow sufficient time for sphere formation and observe them under suitable conditions to accurately assess their quality.
5. Quality of the cell line: Ensure that the E0771 cell line you are using is authentic and properly characterized to ensure reliable results.
Dear Dr. Hend M Nawara
Adding to the above answer by Saif Wahid
Each cell line exhibits a distinct pattern in the process of sphere formation and growth. Initial plating density for spheroid formation will depend on factors such as cell type, duration of growth phase in a spheroidal format, and the desired size of spheroid at the time of assessment. To best evaluate spheroid formation and growth, you may plate cells at different densities of 100, 200, 1,000, 2000, 5,000, and 10,000 cells per well.
There are some points you need to consider.
1. Avoid any clumps. If clumps are present, pass the cell suspension through a 25-gauge needle gently. Clonality is only guaranteed by single cell plating.
2. The cell density is the critical parameter as it influences clonality. Spheres are prone to aggregation due to both intrinsic locomotion and to experimenter-induced movement of dishes. It is important to ensure that spheres are due to proliferation of cells and not due to aggregation. Sphere forming efficiency is influenced by autocrine/paracrine signal released by cells into the medium. Different cell densities affect sphere numbers.
3. Another important factor is that sphere forming assays predominantly detect cells that are poised for, or are actively undergoing proliferation. Quiescent cells may not be capable of forming spheres, either due to intrinsic cell properties or due to lack of additional extrinsic signals needed for their activation in the assay.
Regards,
Malcolm Nobre
From an early age I knew the phrase "Beat their swords into plowshares”, I knew this sculpture well. And I was not alone. And I think that there will come a time when "they shall bind their swords to ploughshares, and their spears to sickles: the people shall not lift up swords against the people, neither shall they learn to fight any more".
Let Us Beat Swords Into Ploughshares
Let Us Beat Swords Into Ploughshares is a bronze sculpture by artist Evgeniy Vuchetich (1908 ¬– 1974). Vuchetich was an esteemed Soviet sculptor and artist well-known for his heroic monuments. In 1959, he was given the “People’s Artist of the USSR” award.
The sculpture depicts the figure of a man, holding a hammer aloft in one hand and a sword in the other hand, hammering the sword into a ploughshare, a tool to till land for crops. This action symbolizes man’s desire to put an end to war and transform tools of destruction into tools to benefit mankind.
The Book of Isaiah contains the following passage: "They shall beat their swords into ploughshares, and their spears into pruning hooks; nation shall not lift up sword against nation, neither shall they learn war any more."
The sculpture was gifted to the United Nations by the USSR on December 4th, 1959. It was presented on behalf of the Soviet Delegation by Vassily V. Kuznetsov, First Deputy Foreign Minister of the USSR, to Secretary-General Dag Hammarskjold who accepted the sculpture on behalf of the United Nations.
https://www.un.org/ungifts/let-us-beat-swords-ploughshares#:~:text=The%20Book%20of%20Isaiah%20contains,they%20learn%20war%20any%20more.%22
Please excuse me, I posted my reply here by mistake. """"Thank you for your understanding and approval. From an early age I knew the phrase "Beat their swords into plowshares” """"
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