A simple and fast method for fixation of cultured cell lines that preserves cellular structures containing gamma-tubulin

Graphical abstract

Non-transfected cells will die within 2-3 days.
3. Add fresh medium supplemented with 100 mg/mL zeocin and 200 mg/mL G418 as deemed suitable.
Passages to new cell culture dishes are not necessary until the 35-mm tissue culture dish is filled to capacity. It takes 2-3 weeks to obtain a stable cell line.
Step 2: cell imaging of live and fixed cells Materials Phosphate-buffered saline (PBS) Freshly made 4% paraformaldehyde and 2% sucrose dissolved in PBS (Psuc buffer); this solution can be stored at À20 C for approximately 2-3 weeks 1:1 methanol, acetone solution (Met/Ac) A felt-tip pen A fluorescence/confocal microscope with a cell incubator equipped with a heating and CO 2 system (necessary for incubations longer than 3 h) Note that the following list includes only necessary non-standard laboratory equipment.
1. Before starting cell imaging, place Met/Ac at À80 C in a freezer and prepare/thaw Psuc buffer. 2. Warm and CO 2 equilibrate the microscope's incubator 30 min before starting the imaging of the cells.
3. Place gTUBULINsh-U2OS-GFP-g-tubulin 334-449 cells in a 35-mm MatTek glass bottom dish positioned in the dish holder of the microscope. 4. Find a suitable cell (Fig. 1A). Most cells in a stable gTUBULINsh-U2OS-GFP-g-tubulin 334-449 cell line form tubular structures containing GFP-g-tubulin 334-449 (Fig. 1A) [2]. 5. Mark the location examined in the dish with a felt-tip pen so that the same position can be found again. An optional approach at this point is to remove the dish from the microscope and then put it back and re-examine it to find the same location again, and this procedure can be repeated until the same cell is found each time. 6. Drawing a mark on the plate may move the dish to some degree; find the chosen cell again and obtain a differential interference contrast (DIC) and a fluorescence (excitation max 488 nm, emission max 509 nm) image (Fig. 1A).
7. Rapidly remove the growth medium from the dish and replace it with 500 mL of Psuc buffer. Fix the cells for 3 min ( Fig. 1A and B) at room temperature. 8. Remove the Psuc buffer and rapidly replaced Psuc buffer with 1 mL À80 C Met/Ac. Immediately thereafter, incubate the dish for 3 min at À80 C in a freezer. 9. Take the dish out of the freezer and remove the Met/Ac. Go to step 11. 10. If a final Met/Ac fixation and permeabilization step is not required, omit steps 7 to 9. Rapidly remove the growth medium from the dish and replace it with 500 mL of Psuc buffer. Fix the cells for 5 min (Fig. 1C and D) at room temperature. 11. Wash the cells twice with 1 mL of PBS. 12. Add 1 mL of PBS. 13. Put the dish containing the fixed cells in the microscope dish holder. Use the pen markings to place the plate in the approximately the same position as previously in the holder and find the previously imaged cell. 14. Search the sample thoroughly by starting at a certain point and moving up and down in the field of view. When lateral movements are necessary, make sure that the new microscope field partially overlaps with the previous one so that no area in the sample is overlooked. Keep searching until the same cell is found (Fig. 1A and C). Obtain a DIC and a fluorescence image (Fig. 1B and D).
Compare the captured images of the fixed cell with the images of the live cell to ensure that the same cell has actually been identified.
Note that in comparison to live cells, the method that better preserves g-tubulin-containing structures in fixed cells is the two-step fixation using paraformaldehyde followed by fixation with methanol/acetone (Fig. 1) [1,3 -5].
Imaging of endogenous g-tubules in fixed cells [1] Step 1: plating cells Materials g-tubules can be found [1].