PCR for sex determination was done as previously described (Kwan et al., 2020 (link)). Briefly, DNA was isolated and PCR was performed using the Platinum Taq DNA Polymerase PCR kit (Catalog #10966–034, Invitrogen, Carlsbad, CA) as per the manufacturer’s instructions. Primers for the Sry gene were forward: 5′-TGGGACTGGTGACAATTGTC-3′, reverse: 5′-GAGTACAGGTGTGCAGCTCT-3′ (Integrated DNA Technologies, Coralville, IA). The bands at 402bp for the Sry amplicon were visualized using the BioRad Molecular Imager Gel Doc XR+ Imaging System (BioRad, Hercules, CA).
Molecular imager gel doc xr imaging system
Manufactured by Bio-Rad
Sourced in United States
The Molecular Imager Gel Doc XR+ Imaging System is a versatile laboratory equipment designed for capturing and analyzing images of electrophoresis gels, blots, and other samples. It features a high-resolution camera, powerful imaging software, and a range of illumination options to provide clear and detailed images for various applications.
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Lab products found in correlation
Rneasy mini kit, by Qiagen (2 mentions)
Seakem le agarose, by Lonza (1 mentions)
Ssofast evagreen supermix kit, by Bio-Rad (1 mentions)
Rnase free dnase 1, by Qiagen (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
F 7100 spectrofluorometer, by Hitachi (1 mentions)
Taq polymerase, by Takara Bio (1 mentions)
Primescript 1st strand cdna synthesis kit, by Takara Bio (1 mentions)
Hybond p, by GE Healthcare (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Moflo high speed sorter, by Agilent Technologies (1 mentions)
Nile red, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Facsaria 2 cell sorter, by BD (1 mentions)
Seakem agarose, by Lonza (1 mentions)
Gelred nucleic acid stain, by Biotium (1 mentions)
1 kb dna ladder, by Merck Group (1 mentions)
22 protocols using molecular imager gel doc xr imaging system
1
Molecular Sex Determination by PCR
2
Colony Formation Assay
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Five hundred cells after transfection were seeded into 6 cm petri dishes with complete medium, and grown up to until visible colonies formed about 2 weeks. Cells were fixed with 4% paraformaldehyde for 30 min and then stained with Coomassie Brilliant Blue (FUDE Biological #FD0022) for 30 min. After washing with deionized distilled water, the dishes were air dried at room temperature. Colonies were counted and photographed with a Molecular Imager® Gel Doc™ XR+ imaging system (BIO-RAD). All assays were repeated three times.
3
Quantitative Viral Plaque Assay
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Plaque assays were performed as described previously, with minor modifications20 (link). Briefly, confluent HeLa or Vero cell monolayers in 10-cm cell culture dishes were washed, inoculated with virus in MEM, and incubated at room temperature for 30 min prior to the addition of 0.45% SeaKem LE agarose (Lonza) in MEM containing 2% fetal bovine serum (FBS). Plates were incubated for 65 h, fixed and stained with 4.4% formaldehyde/0.05% crystal violet. Plaque sizes were quantified from digital images of the plates made with a Bio-Rad Molecular Imager Gel Doc XR+ Imaging System and by subsequent image analysis using MATLAB Image Processing Tool v2.8.
4
Collagen Lattice Contraction Assay
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Three-dimensional collagen lattices were prepared according to the method previously described [55 (link)]. In brief, pDFs’ suspensions (1.0 × 105 cells/400 μL; n = 5 animals; p = 1) were mixed in 200 μL of a solution of rat tail tendon collagen type I (5 mg/mL, Cultrex, R&D Systems, Minneapolis, MN, USA). Next, 500 μL of the mixture was added to each well of a 24-well plate, neutralized with 5 μL of 1 M NaOH, and allowed to polymerize. Gel matrices were then overlaid with 500 μL of CM forms diluted 1:1 in pDFs’ culture medium (see Table 1 ). The ADSC-BM medium mixed with pDFs’ media supplemented with 1% penicillin/streptomycin was used as control. The gels were documented with Molecular Imager® Gel Doc™ XR+ Imaging System (Bio-Rad Laboratories, Inc., Hercules, CA, USA), ImageLab 4.1 software 1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and the areas were measured with ImageJ (SciJava software 1.52a; National Institutes of Health; NIH, Bethesda, MD, USA). The kinetics of contraction was calculated as the percentage of the initial gel area at time 0, which was considered to be 100%.
5
RNA Extraction and qRT-PCR Analysis
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Cells were lysed, and total RNA was extracted using RNeasy Mini kit (Qiagen, Valencia, CA) according to instructions from the manufacturer. Genomic DNA was digested with RNase-free DNase I (Qiagen, Valencia, CA) throughout the RNA extraction process. Five hundred nanograms of RNA was transcribed into cDNA using iScript cDNA synthesis kit (BIO-RAD, Berkeley, CA). Quantitative RT-PCR was performed in triplicates with Ssofast EvaGreen Supermix kit (BIO-RAD, Berkeley, CA). A no-template negative control was included for each primer set and constantly found not to generate any PCR products. The primers used in this experiment are shown in S7 Table . The PCR products were loaded on 2% agarose gel and photographed using Molecular Imager gel-doc XR+imaging system (BIO-RAD, Berkeley, CA) and Image Lab software. For relative expression analysis, a comparative cycle threshold method (ΔΔCT) was used. Briefly, each gene of interest was first normalized against endogenous housekeeping control (β-actin), and then the normalized values were further normalized using the control sample.
6
Highly Sensitive Fluorescence-Based Cas12a Assay
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DNA oligonucleotides (Table 2 ) were synthesized and purified by Sangon Biotech (Shanghai, China). Cas12a protein (Cas12a) was obtained from Guangzhou Meige Biotechnology Co., Ltd. (Guangzhou, China). T4 polynucleotide kinase (T4 PNKP), Lambda exonuclease (λ exo), T7 RNA polymerase, and rNTP were obtained from New England Biolabs (Beijing, China). The water used in the experiment was ultrapure water (18.2 MΩ·cm), and the other reagents were of analytical grade.
Fluorescence spectra were obtained using a Hitachi F-7100 spectrofluorometer (Hitachi, Japan). Gel electrophoresis images were scanned using the Molecular Imager® GelDoc™ XR+ Imaging System (Bio-Rad, Hercules, CA, USA). The water (18.2 MΩ·cm) used in the whole experiment was pretreated with a Milli-Q ultrapure water treatment system (Millipore, Burlington, MA, USA).
Fluorescence spectra were obtained using a Hitachi F-7100 spectrofluorometer (Hitachi, Japan). Gel electrophoresis images were scanned using the Molecular Imager® GelDoc™ XR+ Imaging System (Bio-Rad, Hercules, CA, USA). The water (18.2 MΩ·cm) used in the whole experiment was pretreated with a Milli-Q ultrapure water treatment system (Millipore, Burlington, MA, USA).
7
Native Agarose Gel Electrophoresis
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The samples were run on 1.5% native agarose gel at a constant voltage of 65 V for 90 min at 4 °C. The running buffer was 1 × TAE-Mg2+ buffer. The electrophoresis equipment was a mini-sub cell GT electrophoresis unit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). After electrophoresis, the gel was stained with GelRed nucleic acid dye and scanned with a Molecular Imager Gel Doc XR+ imaging system (Bio-Rad).
8
Immunoblotting of Human IgG1 Antibodies
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Patient sera (1:20 dilution) was added with HRP-labeled mouse anti-human IgG1 antibody (1:3000 dilution). Blots were visualized after staining with diaminobenzidine (DAB).
The results of immunoblotting were analyzed using Molecular Imager¯ Gel Doc™ XR+ Imaging System (Bio-Rad) with Image Lab 4.0.1 software.
The results of immunoblotting were analyzed using Molecular Imager¯ Gel Doc™ XR+ Imaging System (Bio-Rad) with Image Lab 4.0.1 software.
9
Plasmodium Identification by Nested PCR
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Two percent agarose gel electrophoresis of the PCR products was performed followed by GoldView staining and visualized under UV light using Molecular Imager Gel Doc XR+ Imaging System (BioRad, CA, United States). All positive results of Plasmodium-infected cases were confirmed again through direct sequencing of the second round nested-PCR products by the BGI Company (China), using nested primers. Sequencing reads were imported into the EditSeq module of Lasergene-Ver7.11 to construct ssrRNA gene sequences of the nested-PCR products, which were then blasted at https://blast.ncbi.nlm.nih.gov/ . Partial sequences of 18S ribosomal RNA genes of the Togo isolates were aligned with those of other Plasmodium isolates obtained from blast analysis, using the ClustalW method (EMBL-EBI, Hixton and Cambridge, United Kingdom) of MEGA-Ver7.02 to generate a phylogenetic tree based on the neighbor-joining method (Uwase et al., 2020 (link)).
10
Quantitative Gene Expression Analysis
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Total RNA was isolated from cells by using an RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The RNA (2 µg) was transcribed into cDNA using the PrimeScript 1st Strand cDNA Synthesis Kit (Takara, Otsu, Japan). The transcribed product was amplified by polymerase chain reaction (PCR) into a final volume of 50 µl by using the following sense and antisense primers (5'→3'): ICAM-1 sense, GGT GAC GCT GAA TGG GGT TCC; ICAM-1 antisense, GTC CTC ATG GTG GGG CTA TGA CTC; iNOS sense, TCC AAC CTG CAG GTC TTC GAT GC; iNOS antisense, GGA CCA GCC AAA TCC AGT CTG C; IL-1β sense, AAA CAG ATG AAG TGG TCC TTC CAG; IL-1β antisense, TGG AGA ACA CCA CTT GTT GCT CCA; IL-6 sense, AGA GTA GTG AGG AAC AAG CC; IL-6 antisense, TAC ATT TGC CGA AGA GCC CT; IL-8 sense, ACA TGA CTT CCA AGC TGG CCG; IL-8 antisense, TTT ATG AAT TCT CAG CCC TC; and GAPDH sense, CAT GGG GAA GGT GAA GGT C; GAPDH antisense, TGG ACT CCA CGA CGT ACT CA. Amplification was performed using Taq polymerase (Takara, Otsu, Japan). The products were electrophoresed for 30 min at 100 V on 1% agarose gel. Gels were visualized using a Molecular Imager Gel Doc XR imaging system (Bio-Rad Laboratories Inc., Hercules, CA, USA).
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