For serum zinc measurement, blood was collected in 9 ml serum monovettes (Sarstedt) and was centrifuged at 1841xG for 10 min. Subsequently, 1 ml of serum was diluted equally with deionized water in Eppendorf tubes. Serum zinc concentrations were determined by flame atomic absorption spectrometry (AAS) using an AAnalyst 800 (Perkin-Elmer) as previously described [29 (link)].
Aanalyst 800
Manufactured by PerkinElmer
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The AAnalyst 800 is a high-performance atomic absorption spectrometer designed for accurate and reliable elemental analysis. It offers advanced features for the detection and quantification of metals and metalloids in a variety of sample types.
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280 protocols using aanalyst 800
1
Serum Zinc Measurement by AAS
2
Serum Copper Concentration Determination
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Blood samples were collected from the anterior vein on days 1, 20, and 40. These samples were then centrifuged and stored at −20 °C for the measurement of serum Cu. The concentrations of total protein (TP), albumin (ALB), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in the serum were measured using the Drew Trilogy fully automated biochemical analyser. We slaughtered five pigs per group on the 46th day of the formal feeding period. Fresh liver, kidney, muscle, and faeces samples were collected and stored at −80 °C. Tissue samples (0.3 g each) were separately ground into powder, and 0.3 mL serum samples were taken. These were placed into conical flasks, respectively. Subsequently, 2 mL of perchloric acid (HCLO4) and 5 mL of concentrated nitric acid (HNO3) were added. The flasks were then placed on an electric hot-plate and heated for digestion until a small amount of white smoke emerged and the solution became clear and transparent. After cooling, 2.5 mL of 10% HNO3 was added. The solution was transferred to a 25 mL volumetric flask and diluted to the mark with deionized water. The Cu concentrations in the samples were determined by a flame atomic absorption spectrometer (AAnalyst-800, PerkinElmer, Inc., Shelton, CT, USA).
3
Zeolite A for Heavy Metal Adsorption
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Zeolite
A dosages were varied from 0.025 g, 0.05 g, 0.10 g, 0.15 g, and 0.20
g, to 0.25 g for Cd2+ adsorption, and from 0.025 g, 0.05
g, 0.10 g, and 0.15 g, to 0.20 g for Pb2+ adsorption. The
adsorption of Cd2+ and Pb2+ by different dosages
of zeolite A was investigated through a batch experiment using 25
mL of Cd2+ and Pb2+ solutions with an initial
concentration of 50 mg/L, prepared by diluting a 1000 mg/L stock solution
of Cd(NO3)2·4H2O and Pb(NO3)2. This resulted in zeolite A concentrations of
1 g/L, 2 g/L, 4 g/L, 6 g/L, 8 g/L, and 10 g/L for Cd2+ adsorption,
and 1 g/L, 2 g/L, 4 g/L, 6 g/L, and 8 g/L for Pb2+ adsorption
in each 25 mL batch. A contact time of 60 min at 25 °C was maintained
for each adsorption batch. Subsequently, zeolite A was separated using
a paper filter, and the filtrate was collected for analysis of the
residual concentrations of Cd2+ and Pb2+ using
an atomic absorption spectrophotometer (AAS) (AAnalyst 800, PerkinElmer,
USA). The removal efficiency (R%) and adsorption
capacity (q) of Cd2+ and Pb2+ by each dosage of zeolite A were calculated usingeqs 1 and 2 , respectively.
The zeolite A dosage that exhibited the highest adsorption capacity
was selected as the optimal dosage for the further investigation of
Cd2+ and Pb2+ adsorption.
C0 and Ce represent the initial and residual concentrations
(mg/L) of Cd2+ and Pb2+, respectively. V denotes the volume (L) of each adsorption bath, and m refers to the mass (g) of zeolite A.
A dosages were varied from 0.025 g, 0.05 g, 0.10 g, 0.15 g, and 0.20
g, to 0.25 g for Cd2+ adsorption, and from 0.025 g, 0.05
g, 0.10 g, and 0.15 g, to 0.20 g for Pb2+ adsorption. The
adsorption of Cd2+ and Pb2+ by different dosages
of zeolite A was investigated through a batch experiment using 25
mL of Cd2+ and Pb2+ solutions with an initial
concentration of 50 mg/L, prepared by diluting a 1000 mg/L stock solution
of Cd(NO3)2·4H2O and Pb(NO3)2. This resulted in zeolite A concentrations of
1 g/L, 2 g/L, 4 g/L, 6 g/L, 8 g/L, and 10 g/L for Cd2+ adsorption,
and 1 g/L, 2 g/L, 4 g/L, 6 g/L, and 8 g/L for Pb2+ adsorption
in each 25 mL batch. A contact time of 60 min at 25 °C was maintained
for each adsorption batch. Subsequently, zeolite A was separated using
a paper filter, and the filtrate was collected for analysis of the
residual concentrations of Cd2+ and Pb2+ using
an atomic absorption spectrophotometer (AAS) (AAnalyst 800, PerkinElmer,
USA). The removal efficiency (R%) and adsorption
capacity (q) of Cd2+ and Pb2+ by each dosage of zeolite A were calculated using
The zeolite A dosage that exhibited the highest adsorption capacity
was selected as the optimal dosage for the further investigation of
Cd2+ and Pb2+ adsorption.
C0 and Ce represent the initial and residual concentrations
(mg/L) of Cd2+ and Pb2+, respectively. V denotes the volume (L) of each adsorption bath, and m refers to the mass (g) of zeolite A.
4
Nickel Accumulation in Grass Phytoliths
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After 120 days of thinning of grasses, the plants were harvested from the shoot. Shoot samples were washed three times in deionized water and dried in a forced ventilation oven at 65 • C until constant weight, with subsequent weighing to obtain shoot dry weight. A microwave oven (CEM MarsTM 6) with nitric acid (65% v/v-Merck, Rahway, NJ, USA) was used to extract Ni from the shoots of the grasses. Graphite furnace atomic absorption spectrometry (Perkin-Elmer Analyst 800) was used to determine the Ni concentration in the aerial part of grasses. Quality control of Ni analysis used certified reference material (NIST SRM 1573a Tomato leaves) with a recovery rate of 98 ± 2.
The extraction of phytoliths from the shoot of grasses as prepared and separated was performed using the process adapted from [20] (link). The concentration of phytoliths was determined by weighing 10 g of dry mass from the crushed aerial part in a porcelain crucible which was subsequently subjected to calcination at 600 • C in a muffle furnace for six hours. The resulting ashes were transferred to Falcon tubes, where carbonates were removed by the application of 2.5 mL of 1 mol L -1 HCl. The ashes were then purified using 2.5 mL of hydrogen peroxide (H 2 O 2 ) of 30 volume. The residue underwent consecutive washes with distilled water, followed by centrifugation at 300 rad s -1 for five minutes, with the supernatant being discarded. This procedure was repeated in five cycles. The resulting residue (silica phytolith) was dried at 105 • C in a drying oven until a constant weight was achieved. Quantification was conducted through classical gravimetry, employing a precision analytical balance with an accuracy of 0.00001 g. The phytolith concentration results were expressed in g kg -1 of the initially crushed dry mass.
Nickel was extracted from phytoliths using the USEPA 3052 method, with digestion in a microwave oven (CEM MarsTM 6) with H 2 O 2 + HNO 3 + HF and the addition of H 3 BO 3 [18] . Atomic Absorption Spectrometry using a graphite furnace (AAnalyst 800, Perkin-Elmer, Waltham, MA, USA) was used to determine the Ni concentration in the filtered solutions of phytoliths extracted from the shoots of grasses.
The extraction of phytoliths from the shoot of grasses as prepared and separated was performed using the process adapted from [20] (link). The concentration of phytoliths was determined by weighing 10 g of dry mass from the crushed aerial part in a porcelain crucible which was subsequently subjected to calcination at 600 • C in a muffle furnace for six hours. The resulting ashes were transferred to Falcon tubes, where carbonates were removed by the application of 2.5 mL of 1 mol L -1 HCl. The ashes were then purified using 2.5 mL of hydrogen peroxide (H 2 O 2 ) of 30 volume. The residue underwent consecutive washes with distilled water, followed by centrifugation at 300 rad s -1 for five minutes, with the supernatant being discarded. This procedure was repeated in five cycles. The resulting residue (silica phytolith) was dried at 105 • C in a drying oven until a constant weight was achieved. Quantification was conducted through classical gravimetry, employing a precision analytical balance with an accuracy of 0.00001 g. The phytolith concentration results were expressed in g kg -1 of the initially crushed dry mass.
Nickel was extracted from phytoliths using the USEPA 3052 method, with digestion in a microwave oven (CEM MarsTM 6) with H 2 O 2 + HNO 3 + HF and the addition of H 3 BO 3 [18] . Atomic Absorption Spectrometry using a graphite furnace (AAnalyst 800, Perkin-Elmer, Waltham, MA, USA) was used to determine the Ni concentration in the filtered solutions of phytoliths extracted from the shoots of grasses.
5
Urinary Lead and Delta-ALA Quantification
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Morning first (mid-stream) urine samples were collected in sterilized polypropylene containers of 50 mL capacity (Tarson). Samples were coded appropriately to represent the participant name, sample collection date, etc. The collected samples were transported to the laboratory under refrigerated (4°C) conditions and stored at − 20°C till further analysis (for U-Pb), U-ALA was analyzed within 36 hours of sample collection.
U-Pb was measured at 283.31 nm by graphite furnace-atomic absorption spectrometry (Model: AAnalyst 800, Perkin Elmer, USA) as described by Sachin.[46 ] Urine samples were diluted ten times with a 5% (V/V) solution of ultrapure Suprapur grade nitric acid (Merck, Germany) in ultrapure water. A mixed modifier containing 0.1% (W/V) palladium (Merck, Germany) and 0.06% (W/V) magnesium matrix modifier (Merck, Germany) was mixed with diluted urine and the resulting solution was used for U-Pb estimation. A standard stock solution of lead (100 µg/L traceable to SRM from NIST in HNO3) from Merck, Darmstadt, Germany was used. Working standards (0.5, 1, 3, 5, 10, and 15 µg/L) for the calibration curve were prepared from the stock solution. The calibration curve was prepared daily. The method detection limit was calculated using the standard deviation of 10 replicates of a pooled urine sample (i.e., 3*SD). For quality control measures, percentage recovery study and intra/inter-day precision measurement were implemented. Recovery ranged from 83% to 115%. The coefficient of variation (CV%) for intra-day precision was 0.8% for 1 µg/L and 0.5% for 3 and 5 µg/L, while for inter-day precision, CV% was 2.1% for 1 µg/L, 1.6% for 3 µg/L, and 1.2% for 5 µg/L. The method detection limit was 1 µg/L.
U-ALA was determined according to the colorimetric method as described by Tomokuni and Ogata[47 (link)] and Andrade et al.[48 (link)] A urine sample (750 µL) was taken in an Eppendorf tube and centrifuged for 3 minutes at 2000 rpm. The supernatant (500 µL) was taken and vigorously mixed with 500 µL of sodium acetate buffer (pH 4.6) and 67 µL of ethyl acetoacetate. Subsequently, that solution (~1.67 mL) was kept in the water bath at 100°C for 10 min. After cooling at room temperature, 1.5 mL of ethyl acetate was added to that solution and mixed well to centrifuge for 3 minutes (2000 rpm). The upper organic layer (1 mL) of the centrifuged mixture was taken into a separate test tube and mixed with 1 mL of Ehrlich’s reagent. This mixture was kept for 10 min at room temperature. ALA-pyrrole extracted with ethyl acetate developed a cherry red color with Ehrlich’s reagent. The color intensity of the extracted ALA Pyrrole was measured with a UV-visible spectrophotometer (Model: Lambda 45, Perkin Elmer, USA) at 553 nm. Working standards (0.5, 1.0, 2.0, 4.0, 5.0, and 8.0 mg ALA/L) for the calibration curve were prepared daily from the stock solution. Quality control measures such as recovery percentage (92%–100%) were implemented, and the coefficient of variation (CV%) for intra-day precision were 1.2% (0.5 mg/L), 0.7% (4 mg/L), and 0.5% (8 mg/L), and for inter-day precision, CV% was 2% (0.5 mg/L), 1.5% (4 mg/L), and 1% (8 mg/L). The method detection limit was 0.3 mg/L.
U-Pb was measured at 283.31 nm by graphite furnace-atomic absorption spectrometry (Model: AAnalyst 800, Perkin Elmer, USA) as described by Sachin.[46 ] Urine samples were diluted ten times with a 5% (V/V) solution of ultrapure Suprapur grade nitric acid (Merck, Germany) in ultrapure water. A mixed modifier containing 0.1% (W/V) palladium (Merck, Germany) and 0.06% (W/V) magnesium matrix modifier (Merck, Germany) was mixed with diluted urine and the resulting solution was used for U-Pb estimation. A standard stock solution of lead (100 µg/L traceable to SRM from NIST in HNO3) from Merck, Darmstadt, Germany was used. Working standards (0.5, 1, 3, 5, 10, and 15 µg/L) for the calibration curve were prepared from the stock solution. The calibration curve was prepared daily. The method detection limit was calculated using the standard deviation of 10 replicates of a pooled urine sample (i.e., 3*SD). For quality control measures, percentage recovery study and intra/inter-day precision measurement were implemented. Recovery ranged from 83% to 115%. The coefficient of variation (CV%) for intra-day precision was 0.8% for 1 µg/L and 0.5% for 3 and 5 µg/L, while for inter-day precision, CV% was 2.1% for 1 µg/L, 1.6% for 3 µg/L, and 1.2% for 5 µg/L. The method detection limit was 1 µg/L.
U-ALA was determined according to the colorimetric method as described by Tomokuni and Ogata[47 (link)] and Andrade et al.[48 (link)] A urine sample (750 µL) was taken in an Eppendorf tube and centrifuged for 3 minutes at 2000 rpm. The supernatant (500 µL) was taken and vigorously mixed with 500 µL of sodium acetate buffer (pH 4.6) and 67 µL of ethyl acetoacetate. Subsequently, that solution (~1.67 mL) was kept in the water bath at 100°C for 10 min. After cooling at room temperature, 1.5 mL of ethyl acetate was added to that solution and mixed well to centrifuge for 3 minutes (2000 rpm). The upper organic layer (1 mL) of the centrifuged mixture was taken into a separate test tube and mixed with 1 mL of Ehrlich’s reagent. This mixture was kept for 10 min at room temperature. ALA-pyrrole extracted with ethyl acetate developed a cherry red color with Ehrlich’s reagent. The color intensity of the extracted ALA Pyrrole was measured with a UV-visible spectrophotometer (Model: Lambda 45, Perkin Elmer, USA) at 553 nm. Working standards (0.5, 1.0, 2.0, 4.0, 5.0, and 8.0 mg ALA/L) for the calibration curve were prepared daily from the stock solution. Quality control measures such as recovery percentage (92%–100%) were implemented, and the coefficient of variation (CV%) for intra-day precision were 1.2% (0.5 mg/L), 0.7% (4 mg/L), and 0.5% (8 mg/L), and for inter-day precision, CV% was 2% (0.5 mg/L), 1.5% (4 mg/L), and 1% (8 mg/L). The method detection limit was 0.3 mg/L.
6
Pollen-Bearing Flower Elemental Analysis
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The inflorescences were prepared by detaching pollen-bearing flowers from the peduncle. For elemental analysis, we created analytical subsamples. For C, N, and S, a relatively small sample (i.e., 10 mg dry mass) is required for analysis, whereas for all other elements, the analytical subsamples needed to have a minimum of 150 mg dry mass. For this reason, for C, N, and S analyses, it was possible to create analytical subsamples from separate inflorescences, allowing for the collection of 6–10 analytical subsamples from 6 to 10 inflorescences per tree. Subsequently, the residual material, approximately 150 mg dry mass per sample, comprising pooled pollen-bearing flowers (3–5 samples per tree), was used for P, K, Na, Ca, Mg, Cu, Zn, Fe, and Mn analyses. All the analytical subsamples were ground manually using a porcelain mortar and were then freeze-dried. The C, N and S concentrations were measured in 49 analytical subsamples using a Vario EL III automatic CHNS analyser. For all the other elements, the material was digested in a 4:1 solution of nitric acid (70%) and perchloric acid (65%) using a hotplate. After digestion, the analytical subsamples were supplemented with distilled and deionised (Type 1) water (18 mΩ), and the P concentrations were determined by colorimetry (FIA: MLE FIA flow injection analyser). K, Na, Ca, Mg, Cu, Zn, Fe, and Mn concentrations were determined via atomic absorption spectrometry (Perkin Elmer AAnalyst 200 and Perkin Elmer AAnalyst 800) in 24 analytical subsamples. For C, N, P, and S, the results are expressed as a percentage of dry mass, whereas all the other elements are reported as milligrams per kilogram of dry mass. To determine the degree of analytical precision, four blanks were used for each analysis, along with sulphanilic acid as the reference material for the C, N, and S analyses and four different reference materials for P, K, Na, Ca, Mg, Cu, Zn, Fe, and Mn (National Institute of Standards and Technology USA: NIST SRM 1575a – trace elements in pine needles; NIST SRM 1577c – bovine liver; National Research Council of Canada: DOLT-5: Dogfish Liver Certified Reference Material for Trace Metals and other Constituents; and BOVM-1: Bovine Muscle Certified Reference Material for Trace Metals and other Constituents), which were examined with the analytical subsamples.
7
Mineralization of Dispersed Nanosheets
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We used deionized water to
prepare CaCl2·2H2O (99%, Sigma-Aldrich),
NaHCO3 (Sigma-Aldrich, > 99.7%), and Na2CO3 (Merck, 99.9%) solutions. We prepared a CaCl2·2H2O stock solution whose actual concentration was checked by
using flame atomic absorption spectroscopy (PerkinElmer AAnalyst 800).
Carbonate solutions were prepared freshly immediately before the experiments
to ensure that pH did not change because of interactions with CO2 in air.
Different experimental setups were used to
produce single platelet nanocomposites by mineralization of dispersed
nanosheets (Figure 1 a) and to investigate the effects of titrant mixing and subsequent
fluctuations in the spatial distribution of supersaturation on mineralization
reactions. Batch experiments that allowed for consumption of ions
and a decrease in supersaturation during mineralization were employed
in addition to two feedback control setups that continuously replenished
supersaturation via titrant addition, with differences in titrant
mixing procedure and probe sensitivity. In feedback control experiments,
the standard setup was used for the verification of sheet mineralization
and the modified setup allowed for variations in titrant mixing procedures
(Figure 1 b–d).
prepare CaCl2·2H2O (99%, Sigma-Aldrich),
NaHCO3 (Sigma-Aldrich, > 99.7%), and Na2CO3 (Merck, 99.9%) solutions. We prepared a CaCl2·2H2O stock solution whose actual concentration was checked by
using flame atomic absorption spectroscopy (PerkinElmer AAnalyst 800).
Carbonate solutions were prepared freshly immediately before the experiments
to ensure that pH did not change because of interactions with CO2 in air.
Different experimental setups were used to
produce single platelet nanocomposites by mineralization of dispersed
nanosheets (
fluctuations in the spatial distribution of supersaturation on mineralization
reactions. Batch experiments that allowed for consumption of ions
and a decrease in supersaturation during mineralization were employed
in addition to two feedback control setups that continuously replenished
supersaturation via titrant addition, with differences in titrant
mixing procedure and probe sensitivity. In feedback control experiments,
the standard setup was used for the verification of sheet mineralization
and the modified setup allowed for variations in titrant mixing procedures
(
8
Iron Content Determination in Biological Samples
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The non-heme hepatic and placental iron content was determined by acid digestion of the samples at 100 °C for 10 min, followed by colorimetric measurement of an iron-ferrozine complex (absorbance at 560 nm, Beckman DU-68) as described previously [14 ].
A 20 µL sample of colostrum was diluted in 2 ml of boiling Suprapur-grade nitric acid (Merck, Darmstadt, Germany). The total iron concentration was then measured using the graphite furnace atomic absorption spectrophotometry (AAS) technique (AAnalyst 800, Perkin-Elmer, Waltham, MA, USA). Three samples of a standard reference material (197.94 ± 0.65 Fe mg/kg), were analyzed for normalization of the obtained data.
A 20 µL sample of colostrum was diluted in 2 ml of boiling Suprapur-grade nitric acid (Merck, Darmstadt, Germany). The total iron concentration was then measured using the graphite furnace atomic absorption spectrophotometry (AAS) technique (AAnalyst 800, Perkin-Elmer, Waltham, MA, USA). Three samples of a standard reference material (197.94 ± 0.65 Fe mg/kg), were analyzed for normalization of the obtained data.
9
Zinc-Dependent Immune Cell Modulation
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After obtaining informed consent and explaining the nature and potential consequences of the studies, human venous blood was drawn from healthy volunteer donors, anticoagulated with sodium heparin (B. Braun, Melsungen, Germany) and diluted 1:2 in PBS (Sigma-Aldrich, Steinheim, Germany). PBMC were isolated from whole blood using Lymphocytes Separation Media, 1.077 g/ml (Capricorn Scientific, Ebsdorfergrund, Germany). Isolated PBMC were washed in PBS and adjusted to a final concentration of 1 × 106 cells/mL in culture medium with or without Zn2+. The zinc-adequate (ZA) culture medium consisted of RPMI-1640 (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal calf serum (FCS) “Low Endotoxin” (Bio&Sell, Feucht, Germany) 2 mM l -glutamine, 100 U/mL potassium penicillin and 100 U/mL streptomycin sulfate (all from Sigma-Aldrich). To incubate cells in medium without Zn2+, the described medium was treated for 1 h with Chelex® 100 sodium form (Sigma-Aldrich) to chelate all divalent cations. Afterwards, 500 µM CaCl2 and 400 µM MgCl2 (both from Merck, Darmstadt, Germany) were reconstituted and the pH was adjusted back to the level of the culture medium (pH 7.4). Finally, the Zn2+-deficient (ZD) medium was sterile filtered. The Zn2+ depletion was always confirmed by atomic absorption spectrometry using an AAnalyst 800 (Perkin-Elmer, Waltham, USA). For experiments with Zn2+-reconstituted medium (ZR), the Chelex®-treated medium was measured by inductively coupled plasma mass spectrometry (ICP-MSMS) (Agilent 8900 ICP-MSMS, Agilent Technologies, Waldbronn, Germany). The concentration of the metal ions Zn, Ca, Mg, Cu, Fe and Mn were measured in samples that were diluted with nitrogenic acid, using rhodium as an internal standard (Table S1 ). To obtain a ZR-medium all removed metal ions were reconstituted accordingly and the ZR-medium was measured again by ICP-MS.
10
Zinc Deficiency Model in Jurkat Cells
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The human T lymphocyte cell line Jurkat was cultivated at 37 °C in 5% CO2 in a cell culture medium containing RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/mL potassium penicillin,100 µg/mL streptomycin sulfate, 1 mM sodium pyruvate, and 1% 100x nonessential amino acids (all Sigma-Aldrich, St. Louis, MO, USA).
To obtain a zinc deficiency model, Chelex 100 Resin (Sigma-Aldrich, St. Louis, MO, USA) was used. As Mayer et al. [52 (link)] described, Chelex 100 Resin contains paired iminodiacetate ions, which chelate metal ions, especially divalent ions like zinc. The medium was treated with Chelex 100 Resin for one hour. Afterward, 500 µM CaCl2 (Merck, Darmstadt, Germany) and 400 µM MgCl2 (Sigma-Adrich, St. Louis, MO, USA) were readded, and the pH was adjusted to 7.4. The zinc-deficient medium was filter-sterilized, and the zinc concentration was measured by atomic absorption spectrometry (AAnalyst 800, PerkinElmer, Waltham, MA, USA).
For the zinc-supplemented model, 30 µM of ZnSO4 (Sigma-Adrich, St. Louis, MO, USA) was added to the normal cell culture medium.
To obtain a zinc deficiency model, Chelex 100 Resin (Sigma-Aldrich, St. Louis, MO, USA) was used. As Mayer et al. [52 (link)] described, Chelex 100 Resin contains paired iminodiacetate ions, which chelate metal ions, especially divalent ions like zinc. The medium was treated with Chelex 100 Resin for one hour. Afterward, 500 µM CaCl2 (Merck, Darmstadt, Germany) and 400 µM MgCl2 (Sigma-Adrich, St. Louis, MO, USA) were readded, and the pH was adjusted to 7.4. The zinc-deficient medium was filter-sterilized, and the zinc concentration was measured by atomic absorption spectrometry (AAnalyst 800, PerkinElmer, Waltham, MA, USA).
For the zinc-supplemented model, 30 µM of ZnSO4 (Sigma-Adrich, St. Louis, MO, USA) was added to the normal cell culture medium.
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