GC-EVs were analyzed for the expression of CD9, CD81, and Flotillin-1 using imaging flow cytometry (ImageStreamX Amnis Corporation, Seattle, WA, USA) as described [43 (link)]. Briefly, EVs were coupled to previously sonicated aldehyde/sulfate latex beads (1 × 109 particles determined by NTA per 3 μL of beads) for 1 h at RT with agitation followed by ON incubation at 4 °C and blocking with glycine solution with agitation (100 × 10−3 M 30 min; Merck). Next, EVs–beads were immunostained with mouse monoclonal anti-CD9 (sc-59140, Santa Cruz), anti-CD81 (sc-23962, Santa Cruz), and rabbit polyclonal anti-Flotillin-1 (sc-25506, Santa Cruz) for 1 h, followed by incubation with goat anti-rabbit/anti-mouse Alexa Fluor 488 secondary antibodies (Invitrogen, 30 min) and analysis by imaging flow cytometry. For each sample, 100 000 events were acquired at a 40× magnification. Fluorescence of the stained EVs–beads was excited with a 488 argon laser and collected on channel 2 (505–560 nm). A 745 nm laser was activated for side scatter and collected on channel 6, and bright-field images with adjusted intensity were collected on channel 1. Data analysis was performed using the IDEAS software (Amnis Corporation).
Glycine solution
Manufactured by Merck Group
Sourced in Germany, United States, Ireland
Glycine solution is a laboratory product manufactured by Merck Group. It is a clear, colorless liquid containing the amino acid glycine dissolved in water or another suitable solvent. The solution is used as a biochemical reagent and building block in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Sc 23962, by Santa Cruz Biotechnology (1 mentions)
Sc 25506, by Santa Cruz Biotechnology (1 mentions)
Sc 59140, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Tween 20, by Merck Group (1 mentions)
Sulfo nhs, by Merck Group (1 mentions)
Pcdna3 apex2 nes, by Addgene (1 mentions)
Embed 812 resin, by Thermo Fisher Scientific (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Talos l120c, by Thermo Fisher Scientific (1 mentions)
6 m hcl, by Merck Group (1 mentions)
C flex laboratory tubing, by Merck Group (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
Glutaraldehyde solution, by Merck Group (1 mentions)
Tween 80, by Merck Group (1 mentions)
6 protocols using glycine solution
1
Imaging Flow Cytometry of Extracellular Vesicles
2
Synthesis and Characterization of Aminated Graphene
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The aqueous dispersion of the initial GO employed for the synthesis of the aminated graphene was purchased from Graphene Technologies (Moscow, Russia, www.graphtechrus.com (accessed on 24 April 2023)). Formamide (CH3NO), 2-(N-morpholino)ethanesulfonic acid (MES free acid), 1-ethyl-3-carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), Sulfo-NHS, phosphate buffered saline (PBS), Tween 20, sodium hydroxide (NaOH), sodium chloride (NaCl), glycine solution and hydrochloric acid (HCl) were acquired from Merck KGaA (Darmstadt, Germany).
Monoclonal antibodies towards human IgM immunoglobulins were purchased from ImteK, Ltd. (Moscow, Russia). IgM immunoglobulins from human serum (~95% HPLC, buffered aqueous solution) and bovine serum albumin (BSA) were purchased from Merck KGaA (Darmstadt, Germany).
All the organic solvents used in this work were acquired from Vecton Ltd. (Saint-Petersburg, Russia).
All the chemicals were of analytical purity grade, commercially available and used as received without additional purification.
Monoclonal antibodies towards human IgM immunoglobulins were purchased from ImteK, Ltd. (Moscow, Russia). IgM immunoglobulins from human serum (~95% HPLC, buffered aqueous solution) and bovine serum albumin (BSA) were purchased from Merck KGaA (Darmstadt, Germany).
All the organic solvents used in this work were acquired from Vecton Ltd. (Saint-Petersburg, Russia).
All the chemicals were of analytical purity grade, commercially available and used as received without additional purification.
3
Ultrastructural Imaging of APEX2-TM4SF5 Localization
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Cells were transfected with APEX2‐TM4SF5 plasmid (homemade using pcDNA3 APEX2‐NES [#49386, Addgene, Watertown, MA, USA]) for 48 h, fixed with 2% glutaraldehyde and 2% paraformaldehyde solution at 4°C for 16 h, washed 3 times with 100 mmol/L sodium cacodylate buffer (97068, Sigma‐Aldrich), and treated with 20 mmol/L glycine solution (50046, Sigma‐Aldrich). The cells were then stained with freshly diluted 0.5 mg/mL DAB (D12384, Sigma‐Aldrich) in 10 mmol/L H2O2 solution, washed with sodium cacodylate buffer 3 times, fixed using 2% (W/V) osmium tetroxide (75633, Sigma‐Aldrich) for 40 min on ice, rinsed with ice‐cold distilled water, and dehydrated in a graded ethanol series (50%‐100%) for 15 min at each step. The sample was then mixed 1:1 (v/v) with EMBED‐812 resin (50‐980‐446, Thermo Fisher Scientific) and anhydrous ethanol for 1 h. The mixture was incubated overnight in 2:1 (v/v) resin and then exchanged with 100% resin for 2 h before transfer to fresh resin, followed by polymerization at 60°C for 24 h. Embedded cell pellets were cut with a diamond knife into 50 nm sections and imaged on an FEI‐Tecnai G2 Spirit Bio Twin TEM instrument. Images were taken using a 120 kV transmission electron microscope (Talos L120C, FEI, Hillsboro, OR, USA) at the National Instrumentation Center for Environmental Management (NICEM) in Seoul National University (Seoul, S.
4
Synthesis of Gelatin Nanoparticles
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Positively charged gelatin A (Gel A; Bloom number 285) and negatively charged gelatin B (Gel B; Bloom number 247) powders were kindly provided by Rousselot (Rousselot, Ghent, Belgium). Gelatin NPs were made using an acetone-based desolvation method, as previously described [21 (link)]. In brief, gelatin powder was dissolved in demi-water at 5% w/v while stirring at 400 rpm at 40 °C. After dissolution, the pH was adjusted to 2.5 with 6 M HCl (37% w/v fuming, Merck Millipore, Burlington, MA, USA). Thereafter, 135 mL of acetone (Boom, Meppel, The Netherlands) was added at a flow rate of 8 mL min−1 while vigorously stirring (1000 rpm), which induces desolvation of gelatin into spherical NPs. After cooling down, the NPs were crosslinked with 316 µL of 25% glutaraldehyde (Acros Organics, Geel, Belgium) and stirred at 400 rpm overnight at RT. The next day, 100 mL of 100 mM glycine solution (Sigma-Aldrich) was added to capture unreacted glutaraldehyde. NPs were collected by centrifugation (40 min at 16,800× g, 25 °C) and washed twice with demi-water. Afterward, the washed NPs were redispersed in a 30/70% v/v mixture of acetone and demi-water, snap-frozen in liquid nitrogen, and lyophilized for 48 h.
5
Gelatin Microsphere Fabrication Protocol
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Gelatin microemulsions were first fabricated using the microfluidic flow-focusing device. The syringes containing the oil phase (20 wt% Span 80 in mineral oil) and the aqueous phase (5 wt% Type A gelatin solution), respectively, were connected with fluidic ports, using plastic tubing (0.8 mm inner diameter and 1.6 mm outer diameter, C-Flex® laboratory tubing, Sigma). The surrounding temperature was maintained at around 30 °C to prevent the gelatin solution from pre-gelation. The syringes were equipped with syringe pumps (New Era) to control the flow rate of the oil phase at 18.8 μl/min and the aqueous phase at 3 μl/min. The mixture containing gelatin microemulsions and mineral oil was drained out from the chip outlet and was collected in a container. The gelatin microemulsions were maintained at 15 °C and crosslinked to acquire gelatin microspheres (MSs) by adding 10 ml of 5% glutaraldehyde (Sigma) for at least 5 h. MSs were washed with ethanol, cyclohexane, and deionized (DI) water repeatedly, and the excessive aldehyde groups on MSs were blocked in 25 mM glycine solution (Sigma) for 1 h. MSs were lyophilized and kept at room temperature. In order to swell MSs, 10 mg of MSs were immersed in 50 μl of buffers. The mean size of buffer-swollen MSs was observed using an optical microscope and recorded.
6
Fabrication of Gelatin Microspheres for Fibrin Hydrogels
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Fibrin/gelatin hydrogels were produced using a previously described protocol [14] , using a method similar to fibrin/ECM. However, in this particular case, gelatin microspheres [14, 26, 27] were used and incorporated into the fibrin hydrogel as previously described [14] .
Briefly, microspheres were produced by a water-in-oil emulsion method. Gelatin was dissolved in deionised water and added drop-by-drop to 100 ml of olive oil heated to 45°C while being continuously stirred. Gelatin concentration of 11% (w/v) was used in this study.
After 10 minutes, the solution was cooled with additional stirring for 30 minutes, after which 40 ml of acetone was added and left for 1 hour. Formed gelatin microspheres were collected through sieving (50 μm) and repeated washings in acetone. Microspheres were next crosslinked in 100 ml of glutaraldehyde solution (0.1% w/v; Sigma-Aldrich, Ireland) with 100 μl Tween 80 (Sigma-Aldrich, Ireland) for 18 hours while being stirred. Then they were removed from the glutaraldehyde solution and stirred in 100 ml of glycine solution (25 mM, Sigma-Aldrich, Ireland) solution for 1 hour. Microspheres were sieved to a controlled range (50-70 μm), which was used for previously reported release studies [14] . Finally, microspheres were freeze-dried overnight, weighed and sterilized using dehydrothermal treatment [14] .
Briefly, microspheres were produced by a water-in-oil emulsion method. Gelatin was dissolved in deionised water and added drop-by-drop to 100 ml of olive oil heated to 45°C while being continuously stirred. Gelatin concentration of 11% (w/v) was used in this study.
After 10 minutes, the solution was cooled with additional stirring for 30 minutes, after which 40 ml of acetone was added and left for 1 hour. Formed gelatin microspheres were collected through sieving (50 μm) and repeated washings in acetone. Microspheres were next crosslinked in 100 ml of glutaraldehyde solution (0.1% w/v; Sigma-Aldrich, Ireland) with 100 μl Tween 80 (Sigma-Aldrich, Ireland) for 18 hours while being stirred. Then they were removed from the glutaraldehyde solution and stirred in 100 ml of glycine solution (25 mM, Sigma-Aldrich, Ireland) solution for 1 hour. Microspheres were sieved to a controlled range (50-70 μm), which was used for previously reported release studies [14] . Finally, microspheres were freeze-dried overnight, weighed and sterilized using dehydrothermal treatment [14] .
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