Detection of extracellular concentration of procoagulant proteins Tissue factor (TF) and Podoplanin (PDPN) was made by enzyme-linked immunosorbent assays (ELISAs). TF is associated with hypercoagulation in cancer patients [47 (link)]. PDPN is associated with thrombotic disease [48 (link)], and it seems to contribute to cancer progression as a mediator of cancer promoting inflammation [49 (link)]. Extracellular TF was detected and quantified using the Human Tissue Factor ELISA kit (ab220653, abcam, Cambridge, UK). Extracellular PDPN was detected and quantified using the Human Podoplanin ELISA kit (EH375RB: Invitrogen, ThermoFisher Scientific). Prior to the ELISAs, the conditional culture media were concentrated in ultrafiltration centrifuge tubes (UFC8010, Merck, Sigma-Aldrich) at 3220× g for 6 min. After concentrating the samples, the assays were performed following the manufacturer’s protocols. The original concentrations of TF and PDPN in the tested samples were calculated by extrapolating the results measured to the original sample volume.
Ufc8010
Manufactured by Merck Group
The UFC8010 is a laboratory equipment product manufactured by Merck Group. It is a precision instrument designed for laboratory use, but a detailed description of its core function is not available at this time.
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Lab products found in correlation
5 protocols using ufc8010
1
Quantification of Extracellular TF and PDPN
2
Centrifugation and Filtration of Conditioned Media
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For in vitro experiments, CM was subjected to low-speed centrifugation at 2000 rpm for 10 min. The cell-free supernatants were centrifuged at 4000 rpm for 10 min and subjected to filtration with a 0.22 μm polyethersulfone membrane (#SLGPR33RS, Sigma, St Louis, MO, USA). The supernatants were further centrifuged at 10,000×g for 30 min at 4°C to remove remaining cell debris and at 100,000×g (Type 90 Ti Rotor, Beckman, Brea, CA, USA) overnight at 4°C to remove exosomes. For examining the efficacy of CM in vivo, FBS-free CM was condensed by a filter with a cutoff molecular weight of 3 kD, and the condensed CM (50 µl resuspended in PBS) was intravenously injected from the tail vein. To evaluate the effect of nucleic acids on the anti-tumor action of CM, we treated CM with nucleases for digesting DNA and RNA (#PI88700, Thermo Fisher Scientific, Waltham, MA, USA). We also used four filters with different cutoff weights of 3, 10, 30, and 100 kD (#UFC900324, #UFC8010, #UFC8030, #UFC8100, Sigma) and evaluated the anti-tumor efficacy of the size-fractionated CMs.
3
Quantifying 2′3′-cGAMP Hydrolysis Activity
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To determine 2′3′-cGAMP hydrolysis activity of recombinant proteins, 1 μL of each recombinant protein (2 μM) and 1 μL of 2′3′-cGAMP (21 μM) were added to 8 μL of buffer (50 mM HEPES-KOH pH 7.5, 40 mM KCl, 1 mM DTT). Reactions were carried out at 37 °C for 1 h. Then, each mixture was diluted to 3 mL and transferred to a 10 kDa ultrafiltration tube (Millipore, UFC8010), followed by centrifugation at 4000 g for 5 min at 4 °C. After centrifugation, 50 μL of each supernatant was used to assay the amount of 2′3′-cGAMP with the 2′3′-cGAMP ELISA Kit (Cayman, 501700), according to the manufacturer’s instructions. Each test was performed three times. Results were analyzed statistically via two-tailed Student’s t test.
4
Induction and Removal of Protein S-Nitrosylation
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Protein samples (0.5 mg) were diluted with 1 ml of SNO reaction buffer (250 mM HEPES, 1 mM EDTA, 0.1 mM neocuproine, pH 7.7) for each group. To induce SNO proteins, three groups of protein samples were prepared: The control (GSNO-/AA-) group (added with 10 μl of DMSO), the GSNO-/AA+ group (added with 10 μl of DMSO), and the GSNO+/AA+ group (added with 10 μl of GSNO stock solution (10 mM GSNO in DMSO) to obtain final concentration of 0 μM, 0 μM and 100 μM GSNO in the three groups. The protein samples were incubated at 37°C, and shaken well at 800 rpm for 30 min. After incubation, the protein samples were cooled to 4°C to stop the reaction and minimize the changes in mercaptan modification. The excess GSNO was removed by transferring the sample to 4 ml Amicon filters (Millipore, UFC 8010) and conducting buffer exchanges with 4 ml of cold water at 4°C three times. The protein samples were centrifuged at 4000 × g for 15 min after each exchange. The final sample volume was about 50 μl (26 (link)).
5
Isolation and Culture of Murine ECs and MSCs
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ECFCs were established under adherent culture conditions for 21 days in vitro (DIV) from adult murine bone marrow mononuclear cells (Suzuki et al., 2010 (link)). ECFCs were lifted and cultured for another 14 DIV to obtain committed mature ECs. Aliquots of ECFCs and mature ECs were processed for validation of the immunocytochemical phenotypes. A mouse brain endothelioma cell line (bEnd.3 cells, CRL-2299, American Type Culture Collection, Manassas, VA, USA) were cultured according to the manufacturer’s instructions. MSCs were isolated from whole bone marrow cells of adult mice by their adherence to plastic. Neurospheres were generated from embryonic day 14 or adult mouse forebrain striatum cells by the floating culture method in control medium of MHM supplemented with FGF-2 and EGF, as described previously with some modifications (Kase et al., 2019 ).
At the end of the culture period, ECFCs, mature ECs, EC lines, and MSCs were washed and cultured with MHM for another 1 DIV. CM was then collected and filtered. For immunoblot analysis, MHM was made without transferrin. For immunoblotting and in vivo intraventricle infusion, CM were concentrated with Amicon Ultra centrifugal filter devices (UFC9 010, UFC8 010, Millipore) at 2,380 × g for 30 min. Sample volumes were recovered at approximately 45-fold concentration.
At the end of the culture period, ECFCs, mature ECs, EC lines, and MSCs were washed and cultured with MHM for another 1 DIV. CM was then collected and filtered. For immunoblot analysis, MHM was made without transferrin. For immunoblotting and in vivo intraventricle infusion, CM were concentrated with Amicon Ultra centrifugal filter devices (UFC9 010, UFC8 010, Millipore) at 2,380 × g for 30 min. Sample volumes were recovered at approximately 45-fold concentration.
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