ABSTRACT
Preparation of intact, full-size RNA from tissues or cells requires stringent precautions against ubiquitous and rather stable RNases. Fluorescence-activated cell sorting (FACS) usually aims at the isolation of cells according to cell surface markers on living cells, from which RNA can be obtained by standard protocols. The separation of cells according to intracellular immunofluorescence markers, such as intranuclear, intracytoplasmic, or secreted molecules, requires permeation of the cell membrane for the staining antibodies, which is usually achieved by fixation. However, commonly used fixatives such as ethanol, methanol, or formaldehyde do not inactivate RNases completely, thereby hampering the analysis of complete RNA molecules from fixed cells. We report isolation of intact, full-size RNA suitable for Northern blotting from cells that were fixed by 95% ethanol/5% acetic acid containing RNase inhibitors, stained intracellularly, and sorted by FACS.
Subject(s)
Flow Cytometry/methods , RNA, Messenger/isolation & purification , Acetates , Acetic Acid , Animals , Biomarkers , Cell Separation , Cytoplasm/genetics , Ethanol , Fixatives , Fluorescent Antibody Technique , Mice , Molecular Weight , RNA, Messenger/analysis , Spleen/cytology , Staining and LabelingABSTRACT
We have built a cooling device for cell suspensions in flow cytometry that makes use of the Peltier effect (Barnard RD, Thermo electricity in Metals and Alloys, Taylor and Francis, London; Siemens-Z 34:383-88, 1963). The prototype described here is used for cooling collection tubes during long-duration cell sorting and is capable of maintaining a temperature of 2-5 degrees C in a cell suspension of up to 3 ml. In general, Peltier element-based cooling is useful for equilibrating the temperature of small volumes of fluids. Furthermore, Peltier element-based cooling devices are easy to build and handle.
Subject(s)
Cold Temperature , Flow Cytometry/methods , Animals , Electric Conductivity , Flow Cytometry/instrumentation , Mice , ThermodynamicsABSTRACT
We have modified a FACS I by addition of a tunable dye laser and an optical system for fluorescence detection that allows physically independent measurement of green and red immunofluorescence. These modifications are inexpensive and should be applicable to most single-laser systems. By using the modified machine and double-fluorescence activated selection with H-2Kk-specific monoclonal antibodies labelled with FITC or Texas Red, we have isolated from a murine T-cell lymphoma line variant subclones expressing structurally altered histocompatibility class I (H-2Kk) molecules.
