Isolation cells and proteins from a tissue<\/h2>\n<\/span>\n\n\nTissues have connective parts like collagen and ECM components. In the case of adipose tissue from a patient, we have a lot of collagen. The first step is to get rid of the connective tissue. This process is usually done by the digestion of these connective parts by COLLAGENASE enzymes. We are adding to the freshly isolated sample a collagenase cocktail and leave it for a while with regular shaking. After the digestion process is successfully done we got an orange juice like solution, which is the sign that the collagen has been broken down by the collagenase treatment (Figure 1).<\/p>\n\n\n\n![\"\"](\"https:\/\/meddists.com\/learn\/wp-content\/uploads\/2021\/03\/ADIPO-1024x589.png\")
<\/a>Figure 1. Collagenase treatment of Adipose tissue.<\/strong>
(A) Freshly collected sample from a patient. In the Tissue culture flask, the dark solution is the blood, above which the tissue of the adipocytes is orange coloured.
(B) In the first step we need to remove the blood from the sample
(C) After the removal of the blood, we wash the adipose tissue 5-10 times with phosphate-buffered saline solution (PBS)
(D) Collection of the washing solutions
(E) Washed adipose tissue
(F) Result of 5-7 hours of collagenase treatment with continuous shaking, resulted in an orange juice like structure.<\/figcaption><\/figure>\n\n\n\nAfter the collagenase treatment we need to collect the clear cells separated from the connective tissue remnants, which means we have to centrifuge the samples. By the centrifugation we can distuinguish between the cellular components and the non cellular (ECM & connective tissues) parts. By the repeating centrifugation and washing methods we can have even more clear final product. In order to have a clear adipocyte culture for example we need to wash the samples several times until the solution is transparent enough. This is the sign that the RBC-s are eliminated properly for example (Figure 2).<\/p>\n\n\n\n![\"\"](\"https:\/\/meddists.com\/learn\/wp-content\/uploads\/2021\/03\/ADIPO2-1024x645.png\")
<\/a>Figure 2. Centrifugation steps <\/strong>by which we can eliminate the unpleasant part of the tissue sample. On the right part of the picture is a transparent solution under the \u201cfat\u201d amount which means the RBC-s are eliminated.<\/figcaption><\/figure>\n\n\n\nHowever we have a clear sulotion under the orange coloured fat amount it is still a lot of unneccesary components , while we need just the adipocyte population. In order to get the final clear cell population we need to continue the washing steps and the separation method. For that we need to add another \u201cshot\u201d of collagenase treatment to eliminate the remaining connective tissue parts. After the treatment works properly we can have a mixture of adipocytes (fat containing cells) and some RBC-s which stucked between them. The solution is reddish afterwards (Figure 3).<\/p>\n\n\n\n![\"\"](\"https:\/\/meddists.com\/learn\/wp-content\/uploads\/2021\/03\/ADIPO3-1024x328.png\")
<\/a>Figure 3. Separation of the cellular components<\/strong> with centrifugation and another collagenase treatment. In the end, we can have a reddish coloured solution with a pellet at the bottom of the tube. The colour is reddish caused by the RBCs explode by the hypotonic conditions. In the pellet, we can have cells of interest in which we want to investigate the protein of ours.<\/figcaption><\/figure>\n\n\n\nThe pellet contains all of the adipocyte cells. In the next steps we need to destroy the membrane of them and lyse with another set of solutions to obtain the intracellular proteins, where the potential protein of interest could be there.<\/p>\n<\/span>
![\"\"](\"https:\/\/meddists.com\/learn\/wp-content\/uploads\/2021\/03\/ADIPO.png\")
<\/a>(A) Freshly collected sample from a patient. In the Tissue culture flask, the dark solution is the blood, above which the tissue of the adipocytes is orange coloured.
(B) In the first step we need to remove the blood from the sample
(C) After the removal of the blood, we wash the adipose tissue 5-10 times with phosphate-buffered saline solution (PBS)
(D) Collection of the washing solutions
(E) Washed adipose tissue
(F) Result of 5-7 hours of collagenase treatment with continuous shaking, resulted in an orange juice like structure.<\/figcaption><\/figure>\n\n\n\n
After the collagenase treatment we need to collect the clear cells separated from the connective tissue remnants, which means we have to centrifuge the samples. By the centrifugation we can distuinguish between the cellular components and the non cellular (ECM & connective tissues) parts. By the repeating centrifugation and washing methods we can have even more clear final product. In order to have a clear adipocyte culture for example we need to wash the samples several times until the solution is transparent enough. This is the sign that the RBC-s are eliminated properly for example (Figure 2).<\/p>\n\n\n\n However we have a clear sulotion under the orange coloured fat amount it is still a lot of unneccesary components , while we need just the adipocyte population. In order to get the final clear cell population we need to continue the washing steps and the separation method. For that we need to add another \u201cshot\u201d of collagenase treatment to eliminate the remaining connective tissue parts. After the treatment works properly we can have a mixture of adipocytes (fat containing cells) and some RBC-s which stucked between them. The solution is reddish afterwards (Figure 3).<\/p>\n\n\n\n The pellet contains all of the adipocyte cells. In the next steps we need to destroy the membrane of them and lyse with another set of solutions to obtain the intracellular proteins, where the potential protein of interest could be there.<\/p>\n<\/span><\/a><\/a>