Identifying Subcellular Protein Localization with Fluorescent Protein Fusions After Transient Expression in Onion Epidermal Cells - Plant Cell Morphogenesis: Methods and Protocols - page 79

3. Remove the supernatant with pipette and wash three times
with 500 l sterile water. At every wash step, vortex the parti-

cles for about 1 min, let them settle for 1 min, and pellet them
in a microfuge for less than 2 s ( see Note 2 ).
4. Resuspend particles in 500

μ

l sterile 50 % glycerol by vortexing.
Particles are stable at room temperature for at least 1 month.

μ

3.2 Preparation
of Onion Tissue

1. Cut a fresh onion ( see Note 3 ) into quarters. Remove the
innermost leaves since they are usually too highly curved.
2. Gently cut the epidermis on the adaxial (concave) surface into
small strips of approximately 0.5 mm × 2 mm ( see Note 4 ).
3. Using fi ne forceps, peel off the epidermal strips and lay them,
with the outer surface down, in the center of an agar plate.
Collect enough epidermal strips to cover an area of about
3 cm in diameter. Prepare one petri dish per sample.

1. Combine expression plasmids in microfuge tube with a fi nal
volume of 10

3.3 Preparation
of Macrocarriers μ l ( see Note 5 ). Mix well.

2. Add 25 l M17 particles from Subheading 3.1 (thoroughly

suspended by vortexing), 25

μ

μ l 2.5 M MgCl 2 , 5 μ l 200 mM

spermidine.
3. Vortex mixture for 15 min at half-maximal speed. Let particles
settle for 1 min before pelleting them for less than 2 s in a
microcentrifuge ( see Note 2 ).
4. Remove the supernatant without disturbing the pellet. Add
100 l of 70 % ethanol (freshly prepared), wait 30 s, and

remove supernatant ( see Note 6 ).
5. Repeat wash steps three times with 100 % ethanol to remove
residual water.
6. Resuspend particles in 25

μ

l 100 % ethanol. Vortex particles
for at least 1 min. Set pipettor to 15

μ

l and pipette particles up
and down for a few times to break up larger clumps. Vortex
particles again for 1 min.
7. Set down two macrocarrier disks (BioRad) on fi lter paper in an
empty petri dish ( see Note 7 ). Place 8

μ

l of fi nely suspended
particles into the center of each macrocarrier disk and put
entire petri dish in 37 °C incubator for 5 min to evaporate the
ethanol ( see Note 8 ).

μ

1. Turn on particle gun and vacuum pump.
2. Load rupture disk (1,100 psi; BioRad) and securely tighten
holder ( see Note 9 ). Put stopping screen in macrocarrier
assembly. Place the macrocarrier with the particles facing down
in the holder and place on top of macrocarrier assembly. Slide
macrocarrier assembly on top shelf.

3.4 Bombardment