MN in Human Lymphocytes (method description)
Lymphocyte isolation
Lymphocytes were isolated using Ficoll-Paque density gradients. Blood was diluted 1:1 with phosphate buffered saline, and layered onto Ficoll-Paque with ratio of blood+PBS : Ficoll maintained 4:3. The blood was centrifugated at 1800 rpm for 35 min at room temperature. The lymphocyte layer (buffy coat) was removed and washed twice in PBS at 1200 rpm for 10 min each, following the same wash with media RPMI 1640. Cell density was counted with a hemocytometer.
Culture conditions
Lymphocytes were cultured at 37o in a humidified, 5% CO2 incubator in 15 ml conical polystyrene centrifuge tubes. Typically, each culture consisted of an initial density of 1x106 cells in 2 mls of culture medium. Culture medium consisted of RPMI 1640 supplemented with 10% fetal bovine serum 2mM L-glutamine, 100 units/ml penicillin, 100 ug/ml streptomycin and 1.5% phytohemagglutinin.
Micronucleus assay
Cytochalasin B was added to the cultures at 44 hours post initiation as described by Fenech and Morley (1985). Cytochalasin B prevents the cells from completing cytokinesis resulting in the formation of multinucleated cells. Final concentration of cytochalasin B in the cell cultures was 3 mg/ml.
Lymphocytes were spun directly onto glass slides at 72 hrs after culture initiation using a cytocentrifuge (Shandon, Sewickley, PA). The cells would then be spun onto the slide for 10 min at 600 rpm. Slides would be allowed to air dry before methanol fixation at room temperature for 15 min. Slides were stored at -20o C in a sealed box, desiccated, under N2.
Detailed procedures for performing the antikinetochore antibody staining method have been described elsewhere (Eastmond and Tucker, 1989; Yager et al., 1990). Briefly, methanol fixed slides are incubated for 5 min in PBS containing 0.1% Tween 20. Excess fluid is drained from the slide and 40-50 ul of the antikinetochore antibody (Chemicon, Los Angeles, CA) diluted 1:1 with PBS containing 0.2% Tween 20 is applied. The slide is then coverslipped and placed in a humidified chamber at 37oC for 1 h. Following two washes in PBS containing 0.1% Tween 20 for 5 min each, excess fluid is again drained and the slides are covered with 1:50 dilution of fluorescent goat anti-human IgG (Chemacon, Los Angeles, CA) and incubated again for 1 h. Because the fluorescent-labeled antibodies will fade upon exposure to light, this and all subsequent steps should be conducted in yellow light. The slides were rinsed twice in buffer plus 0.1% Tween 20 and counterstained with 4'6-diamidino-2-phenylindole (DAPI) (2 ug/ml) in an antifade solution (Johnson and Nogueira Araujo, 1981).
For the micronucleus test, 1000 binucleate lymphocytes (those that have undergone one mitotic division) were scored, 500 cells from each duplicate culture, for the number of micronuclei. The presence or absence of a kinetochore spot was determined by switching to the fluorescent filter.
Scoring criteria were as follows: 1) cells should have a round or oval appearance with an intact cytoplasm, 2) nuclei should similarly be round or oval with an intact nuclear membrane, 3) only cells having undergone one nuclear division should be scored for the presence of micronuclei, 4) micronuclei should be counted only if they are one third or less the size of the main nuclei, 5) micronuclei should be stained similar to the main nuclei and 6) micronuclei should be clearly separated from the main nuclei. Scoring was performed by two scorers with 10% of slides being rescored. No difference in scoring was found. All questionable micronuclei were additionally assessed by third Cytogeneticsist.
Replicative index (RI), a measure of cell division kinetics, was scored from the same slides scored for micronuclei by counting the percent of cells containing 1, 2 ,3 or more nuclei out of 400 total cells per individual. RI was calculated as follows:
RI = (1x% mononuclear cells) + (2x%binuclear cells) + (3x% tri) + (4x % tetra--------------------------------------------------------------------------------------- |
