GelMA was synthesized as previously reported.47 (link) Gelatin from porcine (Millipore Sigma, Type A, 300 g Bloom), bovine (Millipore Sigma, Type B, 225 g Bloom), or cold-water fish (Millipore Sigma) was dissolved in 1X PBS at 1 g gelatin per 10 mL 1X PBS at 60°C. After gelatin dissolved, temperature was cooled to 50°C and methacrylic anhydride (Millipore Sigma) was added per three reaction conditions. Briefly, these were defined by the ratio of methacrylic anhydride to gram of gelatin: 750 μl·g−1, 375 μl·g−1, and 93.75 μl·g−1. These reaction conditions and subsequent GelMA samples are referred to as ‘High’, ‘Mid’, and ‘Low’ degrees of substitution, respectively. The reaction was continued for 4 hours at 50°C. The reaction was stopped by adding equivalent volume of 1X PBS. The solution was dialyzed in distilled water using dialysis tubing with a molecular weight cutoff of 12-14 kDa (Spectrum) at 40°C for 7 days, with deionized water changed daily. GelMA was frozen at 80°C overnight and lyophilized for 5 days. The final product was stored at 4°C until use.
Type a
Manufactured by Merck Group
Sourced in United States
Type A is a versatile and reliable laboratory equipment designed for a range of scientific applications. It is capable of performing essential functions required in laboratory settings.
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Lab products found in correlation
Heracell 150i, by Thermo Fisher Scientific (3 mentions)
Methacrylic anhydride, by Merck Group (2 mentions)
Matrigel, by Corning (1 mentions)
Type xiv, by Merck Group (1 mentions)
Isophorone diisocyanate, by Merck Group (1 mentions)
Cck 8 kit, by Dojindo Laboratories (1 mentions)
Gelatin, by Merck Group (1 mentions)
Hoechst 33342, by Leagene (1 mentions)
Matrigel basement membrane matrix, by Corning (1 mentions)
Genipin, by Fujifilm (1 mentions)
Pluronic l101, by BASF (1 mentions)
Trypan blue, by Merck Group (1 mentions)
L glutamine lg, by Merck Group (1 mentions)
Streptomycin p s, by Lonza (1 mentions)
Trypsin, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Penicillin, by Lonza (1 mentions)
Porcine gelatin, by Merck Group (1 mentions)
Bovine gelatin, by Merck Group (1 mentions)
Cold water fish skin, by Merck Group (1 mentions)
20 protocols using type a
1
Synthesis and Characterization of GelMA
2
Feeder-free Induced Pluripotent Stem Cell Culture
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Cell culture dishes coated with gelatin or Matrigel® were used for feeder-free cultures. For gelatin coating, culture dishes were incubated with 0.2% gelatin from porcine skin (Type A, MilliporeSigma) for 24 h in a 37°C humidified incubator, solution aspirated and the gelatin film allowed to air dry before use. For Matrigel® (Corning) coating, culture dishes were incubated with Matrigel® (diluted in cold DMEM/F12, 9 µg/cm2) for 2 h in a 37°C humidified incubator, rinsed once with DMEM/F12 before immediate use. To avoid iMEFs when transitioning to feeder-free cultures, biPSC colonies were picked and dissociated into single cells using TrypLE and plated in GMTi medium. Subsequent passages were as described for propagation on iMEFs. Cells were examined and imaged for morphology, growth characteristics and expression of PSC markers. All cultures were maintained in a humidified incubator at 37°C under an atmosphere of 5% CO2.
3
Escherichia coli and Synechocystis cultivation protocol
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Escherichia coli strains DH5α57 (link) and HB101 (harbouring conjugal plasmid pRL443)58 (link) were used for cloning and triparental mating, respectively. All E. coli strains were cultivated on plant-based LB-agar plates or in liquid LB medium (lysogeny broth) at 37 °C. Antibiotics were added to E. coli culture medium to the final concentrations of 20 μg mL−1 spectinomycin (spec), 100 μg mL−1 ampicillin (amp) and 50 μg mL−1 kanamycin (kan), respectively.
Synechocystis sp. PCC 6803 strain GT-U, a derivative of the non-motile, glucose-tolerant strain GT-Kazusa59 (link) was used as final host for all conjugations. For selection and strain maintenance Synechocystis was cultivated on 0.75% [w/v] agar (Type A, Sigma) plates containing TES-buffered (10 mM, pH 8.0) BG-11 mineral medium60 (link). Pre-cultures were grown in TES-buffered BG-11 liquid medium in 6-well polystyrene at 30 °C and 120 rpm. By default, CuSO4 and Co(NO3)2 was not added to any basic BG11 media preparations in this study in order to: (i) minimize uncontrolled Cu2+ induction of PpetE, and to (ii) provide reference data for follow-up studies using Co2+- induction. For contamination control and plasmid maintenance, all cultures of transformed Synechocystis strains were supplied with 20 μg mL−1 spectinomycin and 50 μg mL−1 kanamycin.
Synechocystis sp. PCC 6803 strain GT-U, a derivative of the non-motile, glucose-tolerant strain GT-Kazusa59 (link) was used as final host for all conjugations. For selection and strain maintenance Synechocystis was cultivated on 0.75% [w/v] agar (Type A, Sigma) plates containing TES-buffered (10 mM, pH 8.0) BG-11 mineral medium60 (link). Pre-cultures were grown in TES-buffered BG-11 liquid medium in 6-well polystyrene at 30 °C and 120 rpm. By default, CuSO4 and Co(NO3)2 was not added to any basic BG11 media preparations in this study in order to: (i) minimize uncontrolled Cu2+ induction of PpetE, and to (ii) provide reference data for follow-up studies using Co2+- induction. For contamination control and plasmid maintenance, all cultures of transformed Synechocystis strains were supplied with 20 μg mL−1 spectinomycin and 50 μg mL−1 kanamycin.
4
Vascular Territories Visualization via Pigmented Gelatin Injection
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To visually distinguish vascular territories, arteries were injected with a gelatin aqueous solution colored with dry pigments: red (SP rouge 55, azo pigment, Color Rare, Villenave d’Ornon, France), blue (SP bleu 7, copper-phthalocyanine pigment, Color Rare, Villenave d’Ornon, France), and green (SP vert 8, halogenated copper-phthalocyanine pigment, Color Rare, Villenave d’Ornon, France). We used blue color for the injection of the ACAs, red for the MCAs, and green for the PCAs.
Preparation of injection material consisted of two steps:
Preparation of injection material consisted of two steps:
Dyes were diluted in distilled water (pigment concentrations were 0.5% for blue and red, 0.25% for green). Then, we heated the mixture using a magnetic hotplate stirrer (IKA RH basic 2, Staufen, Germany) to 32 °C and let it cool down to ambient temperature.
Gelatin from porcine skin (gel strength 300 Bloom, Type A, Sigma-Aldrich, St. Louis, MI, USA) was added to obtain a 5% gelatin concentration. We stirred the mixture at the ambient temperature to get a visually uniform distribution and heated it again to 50 °C while stirring. Once the mix was ready, it was transferred to the injection bottles (gas washing Drechsel bottles).
5
HUVEC growth inhibition by NT4 peptide
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HUVEC were plated at a density of 8 x 103 per well in 96-well pre-coated microplates with 0.1% gelatin from porcine skin, type A (Sigma Aldrich) and incubated at 37°C and 5% CO2. Different concentrations of NT4 peptide (50–0.195 μM) were added 24 h after plating. After 24 h, growth inhibition was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. Data was analyzed by nonlinear regression analysis using GraphPaD prism 5.03 software.
6
Alginate-Gelatin Composite Hydrogels
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Composite hydrogels were prepared from alginate and gelatin in six (6) ratios. Gelatin at three different concentrations of 1, 2, and 3 wt% (Type A, gel strength ∼300 g Bloom, Sigma-Aldrich) was dissolved in saline solution (0.9 %w/v sodium chloride in Milli-Q water) at ∼ 55°C, while solutions of 2 and 3 wt% alginic acid sodium salt were prepared from brown algae (medium viscosity, Sigma-Aldrich). The obtained solutions of alginate and gelatin were then mixed to form composite hydrogels with 6 ratios, which were alginate 2 wt%/gelatin 1 wt% (Alg2Gel1), alginate 2 wt%/gelatin 2 wt% (Alg2Gel2), alginate 2 wt%/gelatin 3 wt% (Alg2Gel3), alginate 3 wt%/gelatin 1 wt% (Alg3Gel1), alginate 3 wt%/gelatin 2 wt% (Alg3Gel2), and alginate 3 wt%/gelatin 3 wt% (Alg3Gel3). Also, solutions of alginate or gelation alone, i.e., alginate 2 wt% (Alg2), alginate 3 wt% (Alg3), gelatin 1 wt% (Gel 1), gelatin 2 wt% (Gel 2), and gelatin (Gel 3) were prepared for the subsequent experiments.
7
Screening for Low Manganese Sensitivity
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First, about 15 000 EMS-mutagenized M2 seeds derived from 15 000 M1 seeds were sown onto MGRL medium solidified with 1.2% agar (Type A; Sigma-Aldrich) without Mn supplementation (0 μM Mn). The 0 µM Mn agar plate was calculated to contain 0.47 µM Mn according to Jain et al. (2009) (link). The seeds were grown for 10 d (25 seeds per 140 cm2) and plants with poor growth compared to the wild-type were selected and transplanted to normal Mn conditions (10 μM Mn). At 7 d after transplanting, plants whose growth recovered were selected as mutants. Secondary screening was performed using M3 seeds derived from M2. The seeds were sown in the low- and normal-Mn media (8 seeds per 140 cm2) and after 10–14 d the mutants that showed growth inhibition only in low-Mn conditions were selected as low-Mn-sensitive mutants.
8
Gelatin Preparation for Diffusion
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Gelatins employed in diffusion experiments were prepared as an equal mix of Type A and Type B gelatins (Sigma-Aldrich). The gelatins were cleaned by allowing them to set first in a 10-cm-diameter beaker at a thickness of ∼0.5 cm. After setting, gelatins were soaked with a chosen solution and tilt-rocked for 2–3 d with multiple solution changes. The gelatins were then re-liquified by warming and allowed to set for 10–30 min in pipettes before completing subsequent preparations for experiments.
9
Gelatin Coating for Surface Stiffness
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Glass microscope slides were optionally coated with a layer of gelatin (Type A, Sigma) of thickness 0.5 mm, and kept in air at room temperature for 30 min prior to the experiment. The surface stiffness was adjusted by adding gelatin mass fractions of Cg = [2, 5, 10, 20]% to PBS. PBS without gelatin was used in the soft-surface limit.
10
Isolation of Cardiac Myocytes from Sprague-Dawley Rats
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Cardiac myocytes were isolated from male Sprague-Dawley rats (200-300 g) as previously described [30] (link). The experiments were performed in accordance with the principles for the care and use of experimental animals published by the Korean Food and Drug Administration. The surgical method was approved by the Animal Care and Use Committees of the Chungnam National University (CNU-00992). Briefly, after rats were anesthetized with sodium pentobarbital (150 mg/kg, intraperitoneal injection), the hearts were taken out with thoracotomy. Then, aorta was tied onto the cannular of Langendorff apparatus for retrograde perfusion at 7 ml/min through the aorta (at 36.5°C). The heart was perfused first with Ca 2+ -free Tyrode's solution composed of (in mM) 137 NaCl, 5.4 KCl, 10 HEPES, 1 MgCl 2 , and 10 glucose (pH 7.3) for 3 min, and then with Ca 2+ -free Tyrode's solution containing collagenase (1.4 mg/ml, type A; EC 3.4.24.3; Sigma-Aldrich) and protease (0.14 mg/ml, type XIV; EC 3.4.24.31; Sigma-Aldrich) for about 12 min. Then, the heart was perfused with Tyrode's solution containing 0.2 mM Ca 2+ for further 5 min. The digested heart was then cut and chopped into pieces for further dissociation of myocytes.
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