As a important mineral element, calcium plays a key role in the growth and the quality control of tree fruits such as apple, it can regulate the hardness, sugar content, storage period, and the physiological disorder during storage. Calcium imaging is the most western method to detect dynamic changes in calcium ions in the cytoplasm. Small molecule fluorescent indicators, such as fluo-4/AM have calcium ion-specific selectivity and can be noninvasively loaded by esterification incubation, which is flexible, rapid, and non-cytotoxic.
This protocol will introduce a method for loading small molecule chemical fluorescent reagent fluo-4/AM into plant protoplasts. Protoplast Extraction Add 0.5 milliliter of enzyme solution into a 1.5 milliliter centrifuge tube. Pick a healthy and ripe apple, then slice the pulp into 10 times 5 times 1 cubic milliliter size.
Place the pulp pieces into enzymatic solution, then close the tube. Incubate tube at 28 degrees for one hour. Shacked at 70 rpm in dark.
After that, wash the pulp pieces, aspirate all the enzymatic solution and then add 0.5 milliliter basic solution. Centrifuge at a speed of 300 g for 2 minutes. Protoplasts suspension can be obtained by aspirating the bottom solution.
Small-molecule chemical fluorescence reagent staining. Prepare the fluo-4/AM loading solution with a 2 millimolar fluo-4/AM, 20%ethyl127, and the 10X phosphate-buffered saline in a one to one to two ratio. Add 1 microliter loading solution of fluo-4/AM to 99 microliter protoplast suspension.
The final concentration of fluorescent dyes is 5 micromolar. Mix the solution then close the tube. At the same time, lanthanum iron the calcium or uniqueness of the small molecular chemical fluorescent agent.
Add one microliter or 10 microliter more lanthanum iron and then one microliter loading solution of fluo-4/AM to 98 microliter protoplasts suspension. The final concentration of lanthanum iron is 100 micromolar. Mix the solution then close the tube.
Incubate at 37 degrees for 30 minutes in dark. After that, centrifuge at the speed of 300 G for two minutes. Aspirate to 17 microliters solution and add a 17 microliter basic solution.
Incubate the protoplast suspension at 37 degrees for 30 minutes to completely After that, aspirate 50 microliter protoplasts suspension and drip on to a slide. Observe under fluorescence microscope. Select the 20x the magnification and the GFP channel to observe.
Uniformly adjust the brightness to 0.5. A critical step in this proposed method is to wash the stain in the protoplasts and incubate them for 30 minutes. Insufficient washing causes excessive backer on the fluorescence during observation and incubation allows for better loading or the probe into the cytoplasm.
Protoplasts Viability Assay Take 99 microliter protoplasts suspension after incubating at 37 degrees for 30 minutes. Add 1 ml at the FD solution to protoplasts suspension. Mix the solution by pipetting up and down several times and then close the tube.
Stay at room temperature for five minutes in dark. Prepare the slides and observe them on the fluorescence microscope with GFP channel. We present to you results Following figure one.
We use the enzymatic method all obtaining protoplasts from the pulp. Some protoplasts had a vacuole while others did not. The protoplasts were stained with FD for five minutes and show the florescent in cytoplasm indicating that high temperature, 37 degrees does not affect the viability or protoplasts Figure two.
The protoplasts exhibited no fluorescence in one of the calcium irons fluorescence indicator or it's not loaded into them. Than in fluo-4/AM was loaded into the protoplast, the cytoplast, but not a vacuole became fluorescent. Lanthanum Irons which effectively block calcium channels are the cell membrane were added to the Protoplast fluo-4/AM loading.
At the final concentration of 100 micromolar lanthanum irons significantly reduce the calcium fluorescence intensity. This results further demonstrated that fluo-4/AM effectively stay in the calcium irons in the cytoplast and it show dynamic changes in calcium irons content. Figure three The fluo-4/AM results were analyzed using IMH approved plus 6.0 software fluo-4/AM staining with addition of lanthanum irons significantly reduced the florescence value of cytoplasmic calcium irons.
In this video, were all protoplasts were obtained by enzymatic hydrolysis. We successfully loaded the fluorescent probes into a protoplasts at 37 degrees. More over, the method did not affect the viability of protoplasts.
In summary, the method described here in kind detailed. The dynamic changes in cytoplasmic calcium ions in apple pulp cells thereby providing 10 equal supports for the further purpose of calcium related studies.
