Biochemistry
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Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells
Chapters
Summary May 16th, 2019
Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy.
Transcript
The role of transition metals in mammalian development is largely underestimated. Emerging evidence shows that metal homeostasis continues to change according to the developmental stage of cells and tissues. Moreover, the specific cellular destinations of metals also varietal in development, and of course, it's challenging to identify their location.
To date, different analytical techniques have been developed to quantify metals, but most are expensive or inaccessible. Intensive research is focused to improve metal determinations in a more accessible and efficient manner. Graphite furnace atomic absorption spectroscopy, GF-AAS, has two main advantages.
It provides low limits of detection for zinc, allowing for accurate trace zinc analysis. It is also a technique that does not require high volumes of sample, microliters of solution tend to be enough to carry out the analysis. The combination of these two advantages make GF-AAS ideal to analyze trace elements in low volume samples.
Several human pathological conditions are related to metal misbalances. Some examples are anemia, acrodermatitis enteropathica, and Wilson's and Menkes'diseases. Therefore, it is important to develop efficient and reliable methods to measure the levels of transition metals in biological samples with high sensitivity and accuracy.
GF-AAS is an excellent example on a technology that facilitates a determination of any given metal in normal physiological conditions, and in pathogenic cases. The method is very versatile. It can be applied to many systems and many elements can be analysis using GF-AAS.
The challenges are to establish the method limit of detection in reality, making sure that the samples fall within this limit. Given the fact that the volume of samples is low, there is not a lot of room for testing, so carefully examination of data needs to take place since the very beginning. After the first analysis of samples, we need to establish what's the dilution factor needed to bring the samples to the limits required for quantification.
Be sure that you are able to fractionate the nuclei properly before going to the AAS. Western blots are always good validation prior to the spectroscopic analysis. While GF-AAS requires low volumes, intracellular pools are in low volumes already.
That gives little room for testing. The first measurements need to be carefully done and examined, so sample treatment can be minimized to just one dilution factor. To begin, culture the cells of interest in 55 square centimeter plates.
Use a vacuum trap to aspirate the culture media. Be sure to remove all traces of media. Use ice cold PBS free of calcium and magnesium to rinse the cells from the desired time points, or culture conditions, three times.
Then add one milliliter of ice cold PBS to the plate, and scrape the cells off the plate. Transfer the cell suspensions to a 1.5 milliliter microcentrifuge tube, and keep it on ice. Centrifuge for 10 seconds at 10, 000 times G, and remove the supernatant by aspiration.
If metal analysis of cytoplasmic and nuclear fractions is desired, resuspend the cell pellet in 400 microliters of ice cold PBS containing 0.1%NP-40, a non-ionic detergent. Transfer 100 microliters to a new microcentrifuge tube as the whole cell sample, and store it at minus 20 degrees celsius. To isolate nuclei, use a P1000 micropipette tip to pipette the 400 microliter cell suspension up and down five to 10 times on ice.
Then, extract five microliters of the cell suspension and transfer it to a microscope glass slide. Place the five microliters under a light microscope using a 40 times objective to verify the nuclei integrity. Centrifuge the remaining 395 microliter cell lysate suspension for 10 seconds at 10, 000 times G.Transfer the supernatant containing the cytosolic fraction to a new microcentrifuge tube.
Next, add 500 microliters of the PBS containing 0.1%NP-40 to rinse the pellet with the nuclear fraction, and centrifuge for 10 seconds at 10, 000 times G.Remove the supernatant, and resuspend the pellet containing the nuclei in 100 microliters of the same solution. To lyse the cells, use a Bioruptor to sonicate the whole cell and nuclei containing solution three times, each for five minutes. Proceed to perform quality control of the purity of the fractions by western blot, using antibodies specific for either the nuclear or cytosolic fractions.
First, add an equal volume of concentrated nitric acid of trace metal grade to the whole cell, cytoplasmic and nuclear fractions containing 100, 500 and 100 microliters of cell suspension in 1.5 milliliter microcentrifuge tubes, and place them in a thermal block at 80 degrees celsius to mineralize the cell culture for one hour. After that, take out the tubes from the thermal block and place them in a rack to continue the acid digestion overnight at 20 degrees celsius. Stop the reaction by adding 30%of the sample volume of hydrogen peroxide.
Bring the sample volume to 500 microliters with 18 mega Ohm purified water. Next, in a 15 milliliter Falcon tube dilute analytical grade standard nitric oxide in purified water at 18 mega Ohms to prepare two volume percent nitric acid solution as the blank solution during calibration. Then, use the two volume percent nitric acid solution to dilute a commercially available 1, 000 ppm zinc stock solution with a concentration of 1, 000 parts per billion.
Prepare working standard solutions from the 1, 000 part per million zinc standard solution, the 1, 000 part per billion solution, then dilute it to 5, 8, 10, 15, 20, and 25 ppb directly with 2 volume percent nitric acid solution. Place one milliliter of 0.1%magnesium nitrate matrix modifier into polypropylene sample cups, and then load the cups into the autosampler carousel. Program the atomic absorption spectrometer to automatically add five microliters of the matrix modifier to the zinc standards and the blank.
Set the same optimized condition on the atomic absorption spectrometer for the standard solutions and the samples, with introduction flow rate at 250 milliliters per minute, injection temperature at 20 degrees celsius, and graphite furnace temperature reaching 1, 800 degrees celsius in four step processes. All the standards and samples of a single experiment should be measured at the same time to avoid mistakes due to calibration differences or evaporation. Optimize the lamp C-HCL to a current of 20 amps, wavelength of 213.9 nanometers, and slit of 0.7 nanometers.
Pipette the samples in polypropylene sample cups, and place them into the autosampler carousel. The lower limit of detection of zinc is 0.01 parts per billion. By increasing the concentration of the standards, the upper limit of detection of zinc is determined to be 20 parts per billion.
After a zinc standard curve is obtained, measure the mineralized samples to determine metal content. If the data obtained is beyond the limit of detection, dilute the samples with 18 mega Ohm purified water treated with 0.1%analytical grade nitric acid. In this rapid isolation of nuclei protocol, a representative western blot from differentiating primary myoblasts shows the purity of subcellular fractions.
The chromatin remodeler enzyme Brg1 was used to identify the nuclear fraction, and tubulin was used to identify the cytosolic fraction. This figure shows representative light micrographs for proliferating and differentiated or confluent monolayers of each cell type, and the corresponding zinc content in whole cell extracts, cytosolic and nuclear fractions. All the cell lines analyzed in this study showed zinc concentrations in the nanomolar range.
Differentiated primary myotubes exhibited higher levels of zinc than proliferating cells. A similar subcellular distribution of zinc was detected in the neuroblastoma derived cell line N2A. On the other hand, the established 3T3-L1 cell line exhibited higher levels of zinc when the pre-adipocytes were proliferating than when they were induced or differentiated.
MCF10A cells showed equal levels of zinc between cytosolic and nuclear fractions in proliferating cells. Once MCF10A cells reach confluence, a 40%decrease in whole cell zinc levels was detected, and the metal was found to be most concentrated in the cytosolic fraction. Graphite furnace atomic absorption spectrometry is a highly sensitive technique to measure transition and heavy metals in biological and environmental samples.
Special care and cleanliness in the preparation of subcellular fractions should be taken, as minimal contamination will likely interfere with the analysis, due to the sensitivity of the equipment. Given the low volumes and trace levels of metals in every experiment, it is difficult to think of a better and more accurate technique to measure zinc than GF-AAS. There are no current techniques that can provide the limits of detection and low volume required at the relatively low cost of GF-AAS.
Graphite furnace atomic absorption spectrometry is accurate, sensitive, cost-effective, and accessible. This analytical technique will continue to improve as elemental detection technologies continues to advance. Future application for detection of zinc and other metals by GF-AAS will include organs, tissues obtained from animal models, and biopsies from patients with disease associated with systemic metal imbalances.
Nitric acid is an extremely corrosive acid capable of causing severe chemical burns very rapidly. This chemical can also react violently with certain compounds such as metallic powders. Because of the hazard posed by nitric acid, it's important to take strict safety measures whenever handling nitric acid.
When handling nitric acid we strongly recommend you wear safety chemical glasses, face shield for splash protection, gloves, and approved vapor respiration if ventilation is not adequate.
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