To provide a nonadherent coating for cell patterning, 200 μL of 3% (w/v) molten UltraPure Agarose 1000 (Thermo Fisher Scientific) was added to the central cavity of an acoustic patterning device and allowed to gel by cooling to room temperature. A suspension of fixed, stained chondrocytes was added to an equivalent volume of molten type VII-A agarose, with both solutions mixed at 37 °C. The final cell density was 3 × 107 cells mL–1 and the final agarose concentration was 1.5% (w/v). 200 μL of this solution was added to the central cavity of the acoustic patterning device and a single piezo-transducer pair was driven at a frequency of 6.7 MHz and an amplitude of 20 Vpp. The acoustic field was maintained for 3 min, during which time the solution was cooling to room temperature. After 1 h, the patterned agarose hydrogel was removed from the device for imaging. Low magnification brightfield images were captured with an Olympus BX51 widefield microscope, while high magnification images were obtained using a Leica SP5 inverted confocal fluorescence microscope.
Ultrapure agarose 1000
Manufactured by Thermo Fisher Scientific
Sourced in United States
UltraPure Agarose 1000 is a high-quality agarose product designed for use in various laboratory applications. It is a purified form of agarose, a polysaccharide derived from red algae, with a low gelling temperature and high gel strength. This agarose is suitable for use in gel electrophoresis, DNA and RNA separation, and other applications where a high-purity agarose is required.
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5 protocols using ultrapure agarose 1000
1
Acoustic Patterning of Chondrocyte Hydrogels
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Acoustic Patterning of Chondrocyte Hydrogels
3
Bacterial Predation Assay Protocol
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Bacterial predation was established in laboratory conditions by setting up a predation assay as described in Rombouts et al.19 (link). E. coli and M. xanthus cells were harvested from LB and CYE media, respectively. Next, cells were concentrated for 5 min at room temperature and resuspended in CF medium (10 mM MOPS (pH 7.6), 1 mM KH2PO4, 8 mM MgSO4, 0.02% (NH4)2SO4, 0.2% sodium citrate, and 0.015% bacto casitone peptone). M. xanthus cells were concentrated to an OD600 of 5, E.coli cells to an OD600 of 0.005. 1 µl cell suspensions were spotted at a distance of ~1 mm on CF 1.5% agar pads made with ultrapure agar (UltraPure Agarose 1000, Invitrogen). Predation assays were then placed onto a layer of CF 1.5% agar in a petri dish closed with parafilm to avoid agar pad evaporation and drying. Samples were incubated from 24 h to 48 h at 32 °C to allow M. xanthus cells to invade the E. coli colony. The list of strains used in this study can be found in Supplementary Table 1 .
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PCR Product Gel Extraction and Purification
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The PCR product was run on a 3% agarose gel (Invitrogen UltraPure Agarose-1000, 16550100) at 80 V for 6 hours. The 532– to 538–base pair (bp) PCR product was cut out from the gel and purified with a purification kit (Promega Wizard SV Gel and PCR Clean-Up System, A9281). The eluted product was purified with a DNA purification kit (Zymo Research Genomic DNA Clean and Concentrator-10 D4011), eluting DNA with 10.5 μl of nuclease-free water.
5
Genotyping Fungal Isolates Using ISSR Markers
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Monosporic isolates were grown on V8 medium and incubated at 25°C for 11 days. Harvested mycelium was then dehydrated and macerated in liquid nitrogen. Total DNA was extracted using CTAB (Doyle and Doyle, 1990) , with samples diluted to 10 ng/mL and stored at -20°C.
A total of 12 inter-simple sequence repeat (ISSR) primers (Table 1) were employed in the study (Table 1), with polymerase chain reaction amplifications performedon a PTC-100 thermocycler (MJ Research, Waltham, MA, USA) using the following program: initial denaturation at 94°C for 3 min; 30 cycles of denaturation at 94°C for 40 s, primer annealing at 48° or 50°C (depending on the primer) for 40 s, and extension at 72°C for 1 min; followed by a final extension step at 72°C for 4 min (Williams et al., 1990) . Amplification products were separated by gel electrophoresis in 2.5% ultrapure agarose 1000 (Invitrogen, Carlsbad, CA, USA), treated with 0.5 mg/mL ethidium bromide, and visualized on an ultraviolet transilluminator. The band profiles obtained for each primer were transformed into a binary matrix (presence = '1' and absence = '0') and concatenated as a single multilocus profile for each isolate.
A total of 12 inter-simple sequence repeat (ISSR) primers (Table 1) were employed in the study (Table 1), with polymerase chain reaction amplifications performedon a PTC-100 thermocycler (MJ Research, Waltham, MA, USA) using the following program: initial denaturation at 94°C for 3 min; 30 cycles of denaturation at 94°C for 40 s, primer annealing at 48° or 50°C (depending on the primer) for 40 s, and extension at 72°C for 1 min; followed by a final extension step at 72°C for 4 min (Williams et al., 1990) . Amplification products were separated by gel electrophoresis in 2.5% ultrapure agarose 1000 (Invitrogen, Carlsbad, CA, USA), treated with 0.5 mg/mL ethidium bromide, and visualized on an ultraviolet transilluminator. The band profiles obtained for each primer were transformed into a binary matrix (presence = '1' and absence = '0') and concatenated as a single multilocus profile for each isolate.
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