The overall goal of this procedure is to measure the levels of heme synthesis in mammalian cells by using a specific radioactive precursor of heme synthesis. This is accomplished by first incubating the cells with C 14 labeled five immuno lilin acid, which will be incorporated into heme. The second step is to collect the cells by scraping and then to lice them using heme extraction buffer.
Next dathyl ether is added to extract heme and the ether phase is collected. The final step is to wash the ether phase with hydrochloric acid to separate porphyrins from heme. Ultimately, levels of heme synthesis can be measured in cell lines, and the results show the differential levels of heme synthesis and various cancer cells and HK 2 93 T cells.
The main advantage of this technique over the existing methods like the use of C 14 glycine or iron 59, is that C 14 ELA is very specific and measures the flux of the whole pathway. Additionally, C 14 radiation is very weak compared to iron 59. The implication of this technique extend toward diagnosis and therapy of cancer and neurological diseases because altered he metabolism is associated with various diseases including cancer and neurological diseases.
The seeding co fluency for cells for the heme assay depends on the cell type and their growth rate seed cells in 3.5 centimeter plates so that the co fluency is 80 to 90%on the day of heme measurement. The use of individual plates is recommended because it is easier to handle individual plates on the day before he measurement. Add the radio labeled five immuno leic acid C 14 A LA to the cells.
Since the amount of radio labeled a LA required may vary between cell lines, a minimum amount required for the cell lines under study should be determined beforehand. For most experiments, 0.1 to 0.3 micro curry is sufficient to get a good measurement to each culture. Dish add 0.1 to 0.3, micro curry five A LA, and cold A LA to reach a final concentration of 20, micromolar of total a L.A addition of cold A LA is optional.
Take appropriate precautions to avoid contamination of the experimenter and the surroundings, and follow local radiation safety guidelines for the disposal of all waste. Incubate the cells with radioactive A LA for 16 hours. To begin this procedure, place the cell culture dishes containing the radio labeled cells on ice and take them to the area designated for radioactivity work.
The entire extraction and evaporation process must be performed in an explosion proof hood. Using a pipette aspirate off the medium, wash the cells once with one milliliter of one x cold. PBS aspirate off the PBS and add one milliliter of cold PBS.
Using a rubber policeman, scrape and collect the cells. Transfer the cells to a pre chilled two milliliter micro centrifuge tube. After collecting the cells from all the plates, spin all the tubes at 15, 000 G for one minute at four degrees Celsius.
Aspirate off the PBS and spin. Briefly, remove the residual PBS and resuspend the cells in 100 microliters of PBS vortex to and keep them on ice. Transfer 10 microliters of the cell suspension from each two milliliter tube to a 1.5 milliliter micro centrifuge tube.
Add cell lysis buffer right away and store on ice. This Eloqua will be used later for the measurement of protein concentration. Spin the remaining 90 microliters of cell suspension in the two milliliter tube at 5, 000 G for 30 seconds at four degrees Celsius.
Remove the PBS completely using a 200 microliter pipette tip. Add 300 microliters of heme extraction buffer, or HEB to each tube and vortex to resuspend. The pellet handle only one to two tubes at a time because the pellet should be resuspended quickly without being left in the HEB for too long.
Once HEB is added to all the tubes and the pellets are resuspended vortex for 15 seconds to mix and put on ice for one minute vortex and ice in this manner three times. Next, add 1.2 milliliters of dathyl ether to each tube. Since Dathyl ether runs out of the pipette tips, quickly pipetted up and down once or twice to help hold it in the pipette tip vortex for 30 seconds.
Spin at 15, 000 G for five minutes at four degrees Celsius. Using a one milliliter pipette transfer the top ether phase from each tube to a new micro centrifuge tube. Add 0.5 milliliters of two normal hydrochloric acid to each tube.
A small amount of trian blue has been added to the hydrochloric acid to give it a pale blue color, which will help distinguish the two phases later. Vortex the tubes to mix and spin at 15, 000 G for five minutes at four degrees celsius after centrifugation. The lower phase will be blue and the upper phase will be colorless.
Transfer the upper colorless ether phase to a new tube. Repeat washing the ether phase with hydrochloric acid two more times each time. Collecting the colorless upper phase in a new tube and discarding the lower blue phase.
Transfer the top phase to a scintillation vial. Dry the samples by keeping them in the chemical hood overnight. To evaporate the dathyl ether on the following day.
Add five milliliters of scintillation fluid to each of the dried samples and shake well to mix. Measure the radioactivity by using a scintillation counter. The heme synthesis level is calculated as the amount of radioactivity in picomolar per milligram of total protein.
The protocol demonstrated in this video was used to compare the levels of heme synthesis between normal and cancer lung cells and the absence and presence of a mitochondrial uncoupling.CCCP. In the absence of CCCP, A higher level of heme synthesis was detected in cancer cells. As expected, the levels of hemo synthesis decreased in the presence of CCCP in both normal and cancer cells.
Heme synthesis levels were also calculated in helo cells in the presence and absence of senil acetone or sa SA is a potent and specific inhibitor of the second enzyme in the heme biosynthetic pathway and has been previously shown to inhibit heme synthesis. The results show that the level of heme synthesis decreased by more than tenfold in the presence of sa. To further demonstrate the use of this assay, the levels of heme synthesis were measured and compared in four cancer and HEK 2 9 3 T-cell lines for statistical analysis.
The heme synthesis levels in cancer cells and HT K 2 9 3 T cells were compared to the heme synthesis levels in HCC 4 0 1 7 cells. Asterisks denote a P value less than 0.005 After its development. This technique paved the way for the researchers in the field of cancer to explore the alter heme metabolism for possible cancer diagnosis and therapy.
After watching this video, you should have a good understanding of how to measure the levels of he synthesis in mammalian cells using the specific radioactive precursor of he synthesis, a LA.
