Cell migration and invasion assays of SW620 cells were performed using a modified Boyden chamber approach (ECM508 and ECM554, EMD Millipore), as we described previously.37 (link) Briefly, TFA and cathelicidin peptide (LL-37) were added to the lower chamber. Control-LV- and TUBB3-LV-infected SW620 (2.5 × 104) cells were seeded into the upper chamber and incubated for 7 h (for cell migration) and 24 h (for cell invasion) at 37°C. The FPRL1 antagonist WRW4 and the P2RX7 antagonist KN62 were added to both upper and lower chambers at the beginning of the cell migration experiments. Some of the SW620 cells were pretreated with control inhibitor (YI00199006, QIAGEN) and miR200c-3p inhibitor (YI04100915, QIAGEN) 24 h before the cell migration assay began. The migrated colon cancer cells through the membrane were stained and determined by absorbance at 650 nm.
Yi00199006
Manufactured by Qiagen
Sourced in Germany
YI00199006 is a lab equipment product from Qiagen. It is a device used for the isolation and purification of nucleic acids, such as DNA and RNA, from various biological samples. The product's core function is to facilitate the extraction and purification of these molecules for further analysis or downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Ecm508, by Merck Group (1 mentions)
Red blood cell lysis buffer, by BioLegend (1 mentions)
Total exosome isolation reagent, by Thermo Fisher Scientific (1 mentions)
Qiazol reagent, by Qiagen (1 mentions)
Ym00479902, by Qiagen (1 mentions)
Mircury lna mirna inhibitor, by Qiagen (1 mentions)
Hiperfect transfection reagent, by Qiagen (1 mentions)
Mircury lna mirna mimic, by Qiagen (1 mentions)
3 protocols using yi00199006
1
Colon Cancer Cell Migration and Invasion Assay
2
Isolation and Manipulation of Serum Exosomes in Mice
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Blood samples were collected in tubes containing 50 µL/tube of 0.5% ethylenediaminetetraacetic acid (EDTA) at the time of donor mouse dissection. The blood samples were centrifuged at 10,000 g for 5 min at 4 °C. The supernatant (serum) samples were used for ELISA assays and serum exosomes extraction. The pellets (blood cells) were resuspended in 10 mL 1X red blood cell lysis buffer (#420301, BioLegend) for 10 min, followed by dilution in PBS. The cell suspension was centrifuged at 8,000 rpm for 5 min at 4 °C, and the supernatant was discarded. The immune cell-containing pellets were resuspended and lysed in Qiazol reagent (#79306, Qiagen) for RNA extraction and RT-PCR experiments.
Serum exosomes of donor mice were prepared using total exosome isolation reagent (#4478360, ThermoFisher), and their quantities were then determined by BCA protein assay. The serum exosomes were diluted in PBS and injected into recipient mice intravenously via tail veins (10 µg per mouse).
For inhibition of miRNAs, control (YI00199006), miR-181b-5p (YCI0201288-FZA), and miR-219-5p (YCI0201241-FZA) inhibitors (Qiagen) were dissolved in PBS. The miRNA inhibitors (10 mg/kg in 100 µL/mouse) were injected into the exosome recipient mice subcutaneously under brief isoflurane anesthesia, as recommended by Qiagen.
Serum exosomes of donor mice were prepared using total exosome isolation reagent (#4478360, ThermoFisher), and their quantities were then determined by BCA protein assay. The serum exosomes were diluted in PBS and injected into recipient mice intravenously via tail veins (10 µg per mouse).
For inhibition of miRNAs, control (YI00199006), miR-181b-5p (YCI0201288-FZA), and miR-219-5p (YCI0201241-FZA) inhibitors (Qiagen) were dissolved in PBS. The miRNA inhibitors (10 mg/kg in 100 µL/mouse) were injected into the exosome recipient mice subcutaneously under brief isoflurane anesthesia, as recommended by Qiagen.
3
Transfection of miRNA mimics and inhibitors in hESCs
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Hsa-miR mimics (339173, miRCURY LNA miRNA Mimic) and inhibitors (339121, miRCURY LNA miRNA Inhibitors) were ordered from Qiagen, Hilden, Germany. Additionally, negative control mimics (YM00479902, Negative Control miRCURY LNA miRNA Mimic) and negative control inhibitors (YI00199006, Negative control A), which both have no homology to any known miRNA or mRNA in human, were ordered from Qiagen, Hilden, Germany. The following miRNAs were labeled with a fluorescent 5′-carboxyfluorescein (FAM) tag: hsa-miR-19a mimic + inhibitor, hsa-miR-19b mimic + inhibitor, negative control A mimic, negative control inhibitor. Thirty thousand decidualized or non-decidualized hESCs were cultured in single wells of a 48 wells plate and transfected with 25 nM miRNA mimic or negative control mimic. Alternatively, 30 000 hESCs were transfected with 150 nM miRNA inhibitor or negative control inhibitor. All transfections were performed in D- or C-medium by using HiPerFect Transfection Reagent according to manufacturer’s instructions (301704, Qiagen, Hilden, Germany). After transfection for 24 h, hESCs were washed with PBS and transfection efficiency was determined by calculating the percentage of fluorescently labeled hESCs from the total number of hESCs per image.
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