Preparation of tissues for DNA flow cytometric analysis

Abstract

A method for measuring DNA in tissue cells by flow cytometry utilizing a one step combination nuclear isolation‐DNA fluorochrome staining procedure is described. A variety of cells and tissues, both in vivo and in vitro, was used to illustrate the universal nature of this technique. These included murine bone marrow, liver testicle, sarcoma brain tumor, rat pancreatic islets, human peripheral blood, colon mucosa, colon cancer, sarcoma and brain tumor tissues. A special nuclear isolation medium, which contained either of the DNA fluorochromes, 4′,6‐diamidino‐2 phenylindole‐2 HCl or propidium iodide, was utilized successfully to isolate single suspensions of DNA fluorochrome stained nuclei in a rapid (5–10 min), consistent manner from a variety of tissues and cells. Multiple sampling of the same tissue or comparison between whole tissues and their single cell isolates showed that a representative sample was being obtained.

Solid tumor preparation for flow cytometry using a standard murine model

“…The in vitro examination of individual cells from human solid tumors has been hindered by deficiencies in existing techniques of tumor specimen preparation (6,7,10,15,18,20,22,23,26,27,29,31,(33)(34)(35)(36)(37).…”

“…transplanted tumors. The addition of trypsin permitted Enucleation techniques have been proposed as a Soh-lower concentration of collagenase and more efficient tion, thus circumventing the need for tissue dissociation lysis of intercellular connections, and produced maxi- (21,29,33,34). In the original paper by Vindelov (311, mum cell yields of 1.3-1.4 x 108/g with dye exclusion freeze-thawing JB-1 mouse ascites tumor cell suspen-viabilities of 90%.…”

“…In Thornthwaite's paper, nuclear yields clumping when exposed to alcohol-water fixation solufrom normal C57BL/6J murine liver were shown to vary tions, which tend to precipitate DNA as a gel. from 4.5 to 8.3 x 107/g depending on exposure to NIM, Formaldehyde, gluteraldehyde, paraformaldehyde, but the data was not compared to theoretical yields nor acetic acid, and ethanol fixatives were explored looking was quantitative yields from human solid tumors pre-as end points at cell loss and at quality and stability of sented (29). Petersen (21) determined that the theoreti-DNA histograms produced.…”

“…Three enucleation techniques were performed in triplicate as described in previous reports. In the first technique described by Thornthwaite et al (29), the tumor was minced in nuclear isolation medium (NIM) composed of 0.01M phosphate buffered saline with 1mM CaC12, 0.5mM MgS04-7H20, 0.6% NP40 (N6507 Sigma St. Louis, MO), and bovine serum albumin (Sigma A7888) and left in NIM for 3 min at room temperature. Cells were syringed to remove clumps, and stained with 50pg/ml propidium iodide (Sigma P5264).…”

“…Enucleation techniques have been suggested as a means of circumventing solid tumor dissociation problems (21,29,33,34). These techniques permit the examination of the nuclear components of tumor cells but require the loss of plasma membranes and cytoplasmic features.…”