Ec plan neofluar 63 1.25 oil objective

To tag with green fluorescent protein (GFP) the septin ring as has been previously described (35 (link)), GFP was inserted in frame at the C terminus of CDC10 by using a nourseothricin selection method (36 (link)). Briefly, the GFP-NAT1 cassette was amplified from plasmid pGFP-NAT1 using primers CDC10-GFP-5DR and CDC10-GFP-3DR; transformation of the cassette into THE1-CIp10 and tSEC6 strains was completed using the lithium acetate method, with a 4-h growth step in YPD added after the heat shock step to allow integration and translation of the NAT1 gene before exposing the cells to nourseothricin, as described previously (36 (link)). Transformants were selected on Difco Sabouraud-dextrose agar (BD, Franklin Lakes, NJ) containing 200 μg/ml nourseothricin (Gold Biotechnology, St. Louis, MO). Primers flanking the CDC10 open reading frame (Table 2) were used to screen for transformants carrying the CDC10-GFP allele. DIC and GFP fluorescence images were acquired after induction of filamentation in buffered RPMI for 6 and 24 h in the presence or absence of DOX and in yeast cells after 24 h in YPD with or without DOX. DIC and GFP fluorescence images were acquired using AxioVision 4.7 software (Carl Zeiss AG, Jena, Germany) and a Zeiss EC Plan-NeoFluar 63×/1.25× oil objective (Carl Zeiss AG, Jena, Germany).

Filamentation in liquid medium was assayed at 37°C in RPMI 1640 supplemented with l-glutamine (Cellgro, Manassas, VA) and buffered with 165 mM 3-morpholinopropanesulfonic acid (Sigma, St. Louis, MO) to pH 7.0 (“buffered RPMI 1640”). In brief, buffered RPMI 1640 with or without DOX was inoculated with cells from overnight cultures at a final density of 5 × 10⁶ cells/ml, followed by incubation at 37°C with shaking at 200 rpm. Cells were visualized by DIC microscopy after 24 h by using a Zeiss EC Plan-NeoFluar 63×/1.25× oil objective (Carl Zeiss AG, Jena, Germany).
Filamentation was assayed on solid YPD with 10% (vol/vol) fetal calf serum (FCS), Medium 199 supplemented with l-glutamine (M199), and Spider medium, as described previously (24 (link)). YPD agar was used for embedded colony observation as described previously (32 (link)). All plates were prepared with and without 20 μg/ml doxycycline. Aliquots of 3 μl of cells from overnight cultures were spotted onto agar plates and incubated at 37°C for 24 h. For embedded colony observation, molten YPD agar was cooled to approximately 45°C and cells from overnight cultures were added to a concentration of 20 cells/ml, poured into individual petri dishes, and incubated for 24 h at 30°C. Filamentation was observed with an inverted microscope (Fisher Scientific, Waltham, MA) at 100× and 400× total magnification.