I've tried before, but contamination occurred. I also could not ascertain whether the results of my isolation work was indeed epithelial cells or not. Any suggestion?
Yes. Also you want to minimize exposure of the organ to trypsin as much as possible. Because, trypsin in and of itself can be harmful to cellular membranes. Therefore, I would suggest that you first wash twice with ambient temperature DPBS-Pen/Strep/Fungizone, then once with ambient temperature Ca+2-free / Mg+2-free DPBS--Pen/Strep/Fungizone containing EGTA (rather than EDTA), then digest with ambient temperature trpsin in a solution of Ca+2-free / Mg+2-free DPBS--Pen/Strep/Fungizone-Trypsin with EGTA (rather than EDTA). EGTA is a specific calcium chelator, where as EDTA chelates any divalent cation, i.e., Ca+2, Mg+2, Zn+2, Mn+2, etc. You are only worried about releasing calcium-dependent binding sites and the other divalent cattions are needed for the overall health of the organ's cells, therefore, EGTA is a better choice to remove bound calcium from the tissues. One uses ambient temperature with trypsin to minimize cellular damage.
I would suggest an allternate isolation protocol using a combination of type-I collagenase (Worthington Biochemical, specializing in enzymes) and dispase (R&D). The collagenase will degrade the RGD-binding sites of the cells to the type-I collagen in the extracellular matrix and dispase will degrade basement membranes. Neither enzyme, in my experience has been harmful to the cells themselves. Use the same ewash sequence as stated above, but replace the trypsin with the collagenase/ dispase combination. One change in the protocol is that you can digest at 37C rather than ambient temperature, which is the optimum temperature for enzymatic activity. Your recoveries of viable cells using EGTA in replace of EDTA, collagenase/dispase in replace of trypsin, and 37C instead of ambient temperature, should be significantly higher.
Lastly, you will need a cell surface label for kidney epithelial cells. I would suggest you first try the Developmental Studies Hybridoma Bank (subsidized by NIH). Contact information is http://dshb.biology.uiowa.edu. The bank contains many antibodies to various species and various antigenic epitopes at REASONABLE prices for academic researchers. Look there first, if you cannot find what you are looking for, search on the internet for the antibody to the antigenic epitope of choice and see if someone sells it commercially. If you find a cell surface antibody for kidney epithelial cells, you can them sort with magnetic bead technology (Miltenyi) and get about a 95% pure population of cells. Remember, in everry buffer solution that you use, include Pen/Strep/Fungizone to prevent contamination and perform your digests in closed systems or within a Class-II laminar flow hood.
Yes. Also you want to minimize exposure of the organ to trypsin as much as possible. Because, trypsin in and of itself can be harmful to cellular membranes. Therefore, I would suggest that you first wash twice with ambient temperature DPBS-Pen/Strep/Fungizone, then once with ambient temperature Ca+2-free / Mg+2-free DPBS--Pen/Strep/Fungizone containing EGTA (rather than EDTA), then digest with ambient temperature trpsin in a solution of Ca+2-free / Mg+2-free DPBS--Pen/Strep/Fungizone-Trypsin with EGTA (rather than EDTA). EGTA is a specific calcium chelator, where as EDTA chelates any divalent cation, i.e., Ca+2, Mg+2, Zn+2, Mn+2, etc. You are only worried about releasing calcium-dependent binding sites and the other divalent cattions are needed for the overall health of the organ's cells, therefore, EGTA is a better choice to remove bound calcium from the tissues. One uses ambient temperature with trypsin to minimize cellular damage.
I would suggest an allternate isolation protocol using a combination of type-I collagenase (Worthington Biochemical, specializing in enzymes) and dispase (R&D). The collagenase will degrade the RGD-binding sites of the cells to the type-I collagen in the extracellular matrix and dispase will degrade basement membranes. Neither enzyme, in my experience has been harmful to the cells themselves. Use the same ewash sequence as stated above, but replace the trypsin with the collagenase/ dispase combination. One change in the protocol is that you can digest at 37C rather than ambient temperature, which is the optimum temperature for enzymatic activity. Your recoveries of viable cells using EGTA in replace of EDTA, collagenase/dispase in replace of trypsin, and 37C instead of ambient temperature, should be significantly higher.
Lastly, you will need a cell surface label for kidney epithelial cells. I would suggest you first try the Developmental Studies Hybridoma Bank (subsidized by NIH). Contact information is http://dshb.biology.uiowa.edu. The bank contains many antibodies to various species and various antigenic epitopes at REASONABLE prices for academic researchers. Look there first, if you cannot find what you are looking for, search on the internet for the antibody to the antigenic epitope of choice and see if someone sells it commercially. If you find a cell surface antibody for kidney epithelial cells, you can them sort with magnetic bead technology (Miltenyi) and get about a 95% pure population of cells. Remember, in everry buffer solution that you use, include Pen/Strep/Fungizone to prevent contamination and perform your digests in closed systems or within a Class-II laminar flow hood.
Thank you very much for your suggestion Mr. Henry Young. I'm very appreciate your answer. Now I'll looking for the label first.
excuse me, but may I contact you later if I have any problem sir? Thank you very much.
Yes, you may contact me. My contact information is listed on ResearchGate as well as reprints you can download.
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