LSC Application: Analysis of cytoplasmic vs. nuclear cyclins showing progression through the cell cycle

Progression of cells through the compartments of the cell cycle are regulated by many different sets of nuclear and cytoplasmic proteins. One such protein, Cyclin B1 has been studied by laser scanning cytometry in order to correlate the expression of the protein with DNA content, and the localization of the probe within the cell.
.....The LSC has the ability to automatically and simultaneously measure fluorescence within both the nuclear area and a defined portion of the cytoplasm in order to track movement of proteins like Cyclin B1 across the nuclear membrane. Using CompuSort, one can capture galleries of fluorescence images in order to confirm the localization of these proteins. cell cycle analysis

Hela cells were analyzed by the LSC to determine the expression of Cyclin B1 and DNA content. Cyclin B1 is expressed in G2, but moves from the cytoplasm into the nucleus as cells progress to mitosis. Cyclin B1 was measured in the nuclear contour (a) as well as in a torus in a designated region surrounding the nuclear contour (b). HELA cells: cyclin B1 and DNA content

Galleries of laser scan images relocated by CompuSort. The laser scan image is created by re-scanning each cell from the selected dot plot regions (a and b) and displaying the PMT-derived fluorescence signal for each pixel. The galleries verify the accuracy of the LSC to automatically measure and localize Cyclin B1. Cells on the left were relocated from the nuclear region (a), and cells expressing Cyclin B1 in a cytoplasmic region (b) are on the right.

Sample Preparation
Cell lines grown in microscopic chamber slides were fixed with increasing concentrations of EtOH and allowed to dry prior to staining. Slides were stained with anti-Cyclin B1 (Pharmingen, San Diego) at a 1:20 dilution in PBS containing 1% BSA for 20 min., followed by 5 PBS/BSA washes. FITC-labeled Goat-anti-Mouse (GAM) was used as a developing reagent at a 1:20 dilution. Cells were counterstained by flooding with a solution of propidium iodide (PI, containing RNAse type A 200 µg/ml, Sigma, St. Louis) in a concentration determined to be commensurate with the FITC staining - typically from 0.1 to 1 µg/ml PI. Slides were coverslipped with a 50% glycerin/PBS solution containing an equivalent concentration of PI.
LSC Analysis
The slide was placed on the LSC stage and a region selected for scanning. The slide was scanned using the 40X objective and 5mw Argon laser power. A red fluorescence threshold and a minimum cell size of 100 pixels identified cells for measurement. The Cyclin B1 perimeter measurement was determined to be in a torus from 4 to 20 pixels surrounding the nuclear threshold.