Washed RBC were covalently grafted (derivitized) with methoxypoly(ethylene glycol) succinimidyl valerate (SVA‐mPEG; Laysan Bio Inc. Arab, AL) using different polymer size (2, 5, 10, 20, and 30 kDa) as previously described 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 . RBC suspensions (12% final hematocrit) were prepared in mPEG buffer (50 mM K2HPO4, 105 mM NaCl, pH 8.0), mixed with the activated mPEG solution (final grafting concentrations of 0–4 mM) and incubated for 30 min at room temperature followed by washing (3×) with isotonic saline. The activated polymer species used in this study non‐specifically targets protein lysine residues on the external surface of the RBC 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 . While only small volumes (2–3 mL) of blood were necessary for the described studies, the semi‐automated methodology used is fully scalable for full units of RBC.
Mpeg sva
Manufactured by Laysan Bio
Sourced in United States
The MPEG-SVA is a specialized laboratory instrument designed for the analysis of video and audio data. It provides a standardized interface for the capture, processing, and storage of multimedia content. The core function of the MPEG-SVA is to facilitate the handling and interpretation of multimedia data in a research or analytical setting.
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Lab products found in correlation
Biotin peg sva, by Laysan Bio (13 mentions)
Trolox, by Merck Group (8 mentions)
Glucose oxidase, by Merck Group (8 mentions)
Catalase, by Merck Group (4 mentions)
D glucose, by Merck Group (4 mentions)
Catalase, by Roche (4 mentions)
Biotin peg, by Laysan Bio (3 mentions)
Ms peg 4, by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by New England Biolabs (3 mentions)
Mal peg sva, by Laysan Bio (3 mentions)
3 aminopropyltriethoxysilane, by Thermo Fisher Scientific (2 mentions)
Anti digoxigenin coated polystyrene beads, by Polysciences (2 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
Neutravidin, by Thermo Fisher Scientific (2 mentions)
Milli q water, by Thermo Fisher Scientific (1 mentions)
Methanol, by Merck Group (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
Aptes, by Merck Group (1 mentions)
Tween 20, by Merck Group (1 mentions)
Mw 5000, by Laysan Bio (1 mentions)
25 protocols using mpeg sva
1
PEGylation of Red Blood Cells
2
Activation Strategies for Resting Leukocytes
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The effects of pan T cell activators [i.e., anti-CD3/anti-CD28 and mitogen (phytohemagglutinin; PHA)], alloactivators (i.e., control MLR and camou aged MLR) and secretomes (i.e., SYN, TA1, IA1 and IA2) on the activation of resting leukocytes were compared (Fig. 1). In pan T cell activation, freshly isolated human PBMC were stimulated with anti-human CD3e in the presence of soluble anti-human CD28 for 3 days, or with PHA for 4 days as previously described (Fig. 1A). [4] Alloactivation was conducted in an MLR system with or without succinimidyl valerate activated (SVA) mPEG (Laysan Bio Inc. Arab, AL) for 10 days (Fig. 1B). [4] Effects of alloactivation were compared to untreated resting PBMC. To explore the immunomodulatory effects of alloactivation-secretome-derived therapeutics, cell-free TA1 and IA1 biologics were produced from mPEG-MLR and MLR respectively. Cell secretions from untreated resting PBMC were collected as SYN. [4] A lymphocyte-cancer cell (HeLa) biotherapeutic IA2 was concurrently developed from a HeLa-MLR as previously described. [4] In allo-and secretome activation studies, proliferation and phenotyping of treated PBMC were measured at day 10 (Fig. 1C). For all activation strategies, PBMC miRNA expression pro les were measured at 72 hours post treatment.
3
Silanization and PEGylation of Coverslips
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Quartz coverslips were treated with 100% ethanol and 1 mM KOH. Then, aminosilanization of coverslips was carried out in a 1% (v/v) (3-Aminopropyl)triethoxy silane (Alfa Aesar, A10668, UK) solution in acetone. PEGylation was carried out by incubating mixture of biotinPEG-SVA and mPEG-SVA (Laysan Bio, AL) in the ratio of 1:10 prepared in 0.1 M NaHCO3 solution on the top of silanized coverslip for 3–4 hr. Finally, PEGylated coverslips were stored under dry nitrogen gas at −20°C.
4
Preparation of Single-Molecule Flow Cells
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Single-molecule flow cells were prepared as previously described (Chandradoss et al., 2014 (link); Filius et al., 2020 (link)). In brief, to avoid non-specific binding, quartz slides (G. Finkerbeiner Inc) were acidic piranha etched and passivated twice with polyethylene glycol (PEG). The first round of PEGylation was performed with mPEG-SVA (Laysan Bio) and PEG-biotin (Laysan Bio), followed by a second round of PEGylation with MS(PEG)4 (ThermoFisher). After assembly of a microfluidic chamber, the slides were incubated with 20 μL of 0.1 mg/mL streptavidin (Thermofisher) for 2 minutes. Excess streptavidin was removed with 100 μL T50 (50mM Tris-HCl, pH 8.0, 50 mM NaCl). Next, 50 μL of 75 pM DNA-labeled peptide was added to the microfluidic chamber. After 2 minutes of incubation, unbound peptide and excess Azide-DNA from the earlier click reaction was washed away with 200 μL T50. Then, 50 μL of 10 nM donor labeled imager strands and 100 nM acceptor labeled imager strands in imaging buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.8% glucose, 0.5 mg/mL glucose oxidase (Sigma), 85 ug/mL catalase (Merck) and 1 mM Trolox (Sigma)) was injected. All single-molecule FRET experiments were performed at room temperature (23 ± 2°C).
5
Imaging Aptamer-Bead Interactions by TIRF
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The quartz slides and coverslips, passivated with PEG (a mixture of mPEG-SVA and biotin-PEG-SVA, Laysan Bio) were assembled to form imaging chambers38 (link). For TIRF experiments, 30 pM RNA aptamer construct(s) were immobilized on the surface via biotin–neutravidin interaction. Finally, imaging buffer was added for data acquisition. Unless otherwise mentioned, the imaging buffer comprised of 50 mM Tris-HCl pH 7.5, 0.8% w/v D-glucose [Sigma], 165 U mL−1 glucose oxidase [Sigma], 2170 U mL−1 catalase [Roche], 3 mM Trolox [Sigma], and predetermined amount of KCl/MgCl2.
For integrated fluorescence-force measurements, the imaging chamber was incubated in blocking buffer (10 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mg mL−1 BSA [NEB], 1 mg mL−1 tRNA [Ambion]) for 1 h. The aptamer constructs were then diluted to 10 pM and immobilized on the surface via biotin–neutravidin interaction. Subsequently, 1 μM anti-digoxigenin coated polystyrene beads (Polysciences), diluted in a buffer containing 10 mM Tris-HCl pH 7.5 and 50 mM NaCl, were added to the imaging chamber and incubated for 30 min. Finally, data were acquired in the imaging buffer.
For integrated fluorescence-force measurements, the imaging chamber was incubated in blocking buffer (10 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mg mL−1 BSA [NEB], 1 mg mL−1 tRNA [Ambion]) for 1 h. The aptamer constructs were then diluted to 10 pM and immobilized on the surface via biotin–neutravidin interaction. Subsequently, 1 μM anti-digoxigenin coated polystyrene beads (Polysciences), diluted in a buffer containing 10 mM Tris-HCl pH 7.5 and 50 mM NaCl, were added to the imaging chamber and incubated for 30 min. Finally, data were acquired in the imaging buffer.
6
PEGylation of Recombinant Human MG53
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PEGylation is a well-established method for increasing the circulating half-life of therapeutic proteins [39 (link)]. We conducted a study with mPEGSVA (purchased from LaySan Bio, Inc., Arab, AL, USA) modification of rhMG53. An amount of 40 mg rhMG53 protein was dissolved in PBS (pH = 8) solution at 1 mg/mL concentration at 4 °C. Then, 40 mg PEG-SVA was added into the rhMG53 solution and mixed gently. The reaction of PEGylation was carried out at 4 °C overnight. Un-conjugated PEG–SVA was filtered out by Amicon 30 ultrafilter with PBS. PEG–rhMG53 was stored at −20 °C for long-term storage or 4 °C for short-term usage and to avoid the freeze–thaw cycle.
7
Biofunctionalization of Quartz Slides
8
Micropatterning Fibrinogen on Functionalized Coverslips
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Glass coverslips were exposed to an air plasma (0.2 mbar, ZEPTO, Diener) for 1 min, coated with 0.1% poly-L-lysine (PLL) for 30 min, washed 5 times with ddH2O, and let to dry. The dried cover slides were passivated with freshly prepared 100 mM HEPES buffer (pH 8.2) containing 100 µg/mL mPEG-SVA (Laysan Bio, Arab, AL, USA) and stored overnight at 4 °C. Residual mPEG-SVA solution was removed by washing the slides in ddH2O, and the slides were let to dry. Next, a polydimethylsiloxane stencil (Alvéole) was placed on the dried cover slides and 2.8 μL of a photosensitizer solution (consisting of 0.8 µL Product of Liaison for Protein Patterning (PLPP, Alvéole) and 2 μL MQ-H2O) were pipetted into each stencil well. The cover slides were then dried on a heat plate for 8 min at 50 °C. Micropatterns were introduced via maskless photopatterning (PRIMO, Alvéole) with a UV laser power of 30 mJ/mm2. After washing with ddH2O and PBS, the micropatterns were coated with AlexaFluor 546-labeled human fibrinogen (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA, 100 μg/mL in PBS) for 5 min and washed 4× with PBS. Slides were stored at 4 °C in humidified conditions until use.
9
Immobilization of Nucleosomes for Single-Molecule Studies
10
Functionalized Coverslips for Biomolecular Imaging
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Coverslips were soaked in piranha solution (25% H2O2 and 75% concentrated H2SO4) and sonicated for 1 h, followed by multiple rinses in water (Thermo Fisher Scientific, molecular-biology grade) and acetone (Thermo Fisher Scientific, HPLC grade). Dry and clean coverslips were then treated with Vectabond/acetone (1% v/v) (Vector Labs) solution for 5 min and then rinsed with water and left in a dried state until used. In order to prevent non-specific adsorption of biomolecules onto the glass surface, coverslips were functionalized prior to use with a mixture of poly(ethylene glycol) succinimidyl valerate, MW 5000 (mPEG-SVA) and biotin-PEG-SVA at a ratio of 99:1 (w/w) (Laysan Bio) in 0.1 M sodium bicarbonate (Thermo Fisher Scientific) for 3 h18 (link). Excess PEG was rinsed with water, and the coverslips were dried under a N2 stream. Imaging chambers (~5 μL) were constructed by pressing a polycarbonate film (Grace Bio-Labs) with an adhesive gasket onto a PEG-coated coverslip. Two silicone connectors glued onto the pre-drilled holes of the film served as inlet and outlet ports. The surface was incubated with 7 μL of a 2 mg/ml neutravidin solution (Thermo Fisher Scientific). Excess neutravidin was then washed off with 100 μL of 1X PBS buffer.
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