An Octet RED96e instrument (FortéBio, Sartorius AG) was used to perform binding experiments of the antibodies to soluble murine PD-1 monoFc fusion protein. The murine PD-1 fusion protein was biotinylated using EZ-Link sulfo-NHS-LC-Biotin (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, the biotin-conjugate was added to protein in 0.1 M sodium phosphate buffer at a ratio of 1:5 molar excess. The reaction proceeded at room temperature with continuous rocking and was stopped with 10x tris-buffered saline after 30 minutes. Any remaining unbound biotin was removed by Amicon® Ultra-15 centrifugal filters (Millipore Sigma) or Zeba Spin Desalting Columns (Thermo Fisher Scientific), and the protein was buffer exchanged to PBS (Corning). The biotinylated protein was then loaded onto SA sensors at 2 μg/mL for 60 seconds. These sensors were then first dipped into PBS (Corning) for 60 s to establish a baseline and then exposed to each antibody solution (varying concentrations) for 600 seconds to measure association. The sensors were then returned to PBS (Corning) for 1800 seconds for dissociation. Curve fitting to calculate apparent KD was performed using the Octet System Data Analysis software (FortéBio, Sartorius AG).
Phosphate buffered saline (pbs)
Manufactured by Corning
Sourced in United States, China, Japan
PBS is a buffered saline solution commonly used in various laboratory applications. It is a versatile and widely-used buffer that helps maintain a stable pH environment for biological samples and procedures. The core function of PBS is to provide a physiologically compatible environment for cells, proteins, and other biomolecules.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (44 mentions)
Bovine serum albumin (bsa), by Merck Group (24 mentions)
Penicillin streptomycin, by Corning (22 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (18 mentions)
Dulbecco modified eagle medium (dmem), by Corning (16 mentions)
Trypsin, by Corning (14 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (14 mentions)
Fetal bovine serum (fbs), by Corning (14 mentions)
Triton x 100, by Merck Group (13 mentions)
Matrigel, by Corning (11 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (10 mentions)
Tween 20, by Merck Group (10 mentions)
Hepes, by Thermo Fisher Scientific (9 mentions)
Rpmi 1640, by Corning (8 mentions)
Trypsin edta, by Thermo Fisher Scientific (8 mentions)
Penicillin, by Thermo Fisher Scientific (8 mentions)
Penicillin, by Merck Group (8 mentions)
Streptomycin, by Merck Group (8 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (7 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (7 mentions)
594 protocols using phosphate buffered saline (pbs)
1
Antibody Binding Kinetics to Murine PD-1
2
Affinity Measurement of 4-1BB Receptor Binding
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96 well plates precoated with rat collagen I (Gibco) were blocked overnight with PBSTA (PBS (Corning) + 2% w/v BSA (Sigma Aldrich) + 0.05% v/v Tween-20 (Millipore Sigma)) at 4 °C. After washing with 3 times PBST (PBS (Corning) + 0.05% v/v Tween-20 (Millipore Sigma)) and 3 times with PBS (Corning), ɑ4-1BB and ɑ4-1BB-LAIR were incubated in PBSTA overnight at 4 °C while shaking. Wells were washed 3 times with PBST and 3 times with PBS and then incubated with goat αmIgG1-Horseradish peroxidase (HRP) (1:2000, Abcam) in PBSTA for 1 h at RT while shaking. Wells were again washed 3 times with PBST and 3 times with PBS and then 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher) was added for 5–15 min, followed by 1 M sulfuric acid to quench the reaction. Absorbance at 450 nm (using absorbance at 570 nm as a reference) was measured on an Infinite M200 microplate reader (Tecan). Binding curves were generated with GraphPad Prism software V9. KD values were calculated using a nonlinear regression fit for one site total binding with no non-specificity and curves were normalized to the Bmax values.
3
CHO-K1 Cell Monolayer Formation and Microscopy Analysis
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The Chinese Hamster Ovary (CHO-K1) cell monolayer was formed on a polyethylene terephthalate (PET) membrane with a porosity of 1 μm (Cell Culture Insert, Becton Dickinson, NJ, USA). Cells were incubated at 37 °C in an atmosphere of 5% carbon dioxide for 48 h. The cell monolayer was washed in phosphate-buffered saline (PBS; Corning, New York, NY, USA) and examined in terms of PET membrane coverage by CHO-K1 cells using ImageJ software [28 (link)] or Giemsa staining before/after a diffusion analysis by laser interferometry.
Images of monolayers were obtained using optical microscopy and were converted into gray-scale digital images (1 denotes black, and 256 denotes white). Using ImageJ software [28 (link)], the degree of PET membrane coverage by cells (confluence) was estimated at around 95%. The CHO-K1 cells forming a monolayer on the PET membrane were washed with PBS (Corning) and fixed in 100% methanol (Sigma–Aldrich, St. Louis, MO, USA) for 5 min. Next, the cells were incubated with a 5% solution of Giemsa in Sorensen buffer (Sigma–Aldrich, St. Louis, MO, USA) for 20 min. Slides were rinsed in water and dried, and images were captured using an A1R inverted microscope (Nikon, Tokyo, Japan) at a 100× magnification.
Images of monolayers were obtained using optical microscopy and were converted into gray-scale digital images (1 denotes black, and 256 denotes white). Using ImageJ software [28 (link)], the degree of PET membrane coverage by cells (confluence) was estimated at around 95%. The CHO-K1 cells forming a monolayer on the PET membrane were washed with PBS (Corning) and fixed in 100% methanol (Sigma–Aldrich, St. Louis, MO, USA) for 5 min. Next, the cells were incubated with a 5% solution of Giemsa in Sorensen buffer (Sigma–Aldrich, St. Louis, MO, USA) for 20 min. Slides were rinsed in water and dried, and images were captured using an A1R inverted microscope (Nikon, Tokyo, Japan) at a 100× magnification.
4
Analyzing MS13 Impact on Prostate Cancer Cells
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In order to examine the gene expression profiling affected by MS13 on the prostate cancer cell lines, both DU 145 and PC-3 cells were seeded at the density of 1 × 107 cells in T75 cm2 flasks (Nunc) and incubated overnight to allow cells attachment at 37 °C in a humidified incubator with 5% CO2. The cells were incubated with 16 μM of MS13 for 24 h duration based on the EC50 values. Meanwhile, the untreated cells received only media with only DMSO. After 24 h treatment, the treated cells were harvested by pre-warmed accutase® Cell Detachment Solution (Innovative Cell Technologies, San Diego, CA, United States), washed with 2 ml of 1× PBS (Corning®) and centrifuged twice to obtain a fresh clean pellet. Then, the fresh pellet was re-suspended in 1,000 ml of 1x PBS (Corning®), kept on ice and further to RNA extraction. The experiment was performed in three biological replicates.
5
PD-1 Binding Affinity Assay
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Ninety-six-well plates were coated with soluble murine PD-1 monoFc fusion protein overnight at 4°C. The coated plates were then blocked at room temperature for at least 2 hours with PBS (Corning) with 10 mg/mL BSA (Thermo Fisher Scientific) and 0.05% vol/vol Tween-20 (Millipore Sigma). The plates were then incubated overnight at 37°C with various concentrations of antibodies in PBSTA (PBS (Corning) with 1 mg/mL BSA (Thermo Fisher Scientific) and 0.05% vol/vol Tween-20 (Millipore Sigma)). Wells were washed with PBSTA four times and then incubated with horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific) at a 1:3000 dilution in PBSTA for 1 hour at 37°C. Wells were washed again four times with PBSTA, and then 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added for 5–10 min followed by 1 M sulfuric acid to stop the chromogenic reaction. Absorbance at 450 nm (corrected with a reference absorbance at 570 nm) was measured on an Infinite M200 microplate reader (Tecan).
6
Isolation of CD4+ T Cells from Infant and Adult Mice
7
Culturing STS26T Cells for Sciatic Nerve Injection
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The STS26T cell line was cultured with complete DMEM media supplemented with 10% FBS until 80%-90% confluency. Cells were then washed with 10 ml of phosphate-buffered saline (PBS; Corning), and 0.5% trypsin-EDTA 10× (Gibco by Life Technologies) was diluted to 1× with PBS. One milliliter of 1× trypsin was then incubated with the cells at 37°C for 1 minute to detach the cells from the culture flask. The cells were resuspended in DMEM media with 10% FBS and centrifuged at 2400g (IEC Centra CL2) for 2.5 minutes. The media was aspirated, and the cells were washed again with media and centrifuged at 2400g for 2.5 minutes. The media was aspirated again. The cells were subsequently resuspended in 1 ml of media and counted manually. The cells were then diluted to 50,000 cells/5 ml of media. This cell resuspension was then aspirated into a 30-gauge Hamilton syringe and used for injection into the mouse sciatic nerve.
8
Isolation and Enumeration of Immune Cells
9
Optimized RNA Encapsulation in Lipid Nanoparticles
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RNA was packaged into LNPs using a NanoAssemblr microfluidic system (Precision Nanosystems, Vancouver, Canada) according to manufacturer instructions. Briefly, LNP formulations were formed by injecting 12.5 mM of the lipid solution and 0.173 μg/μl of RNA in formulation buffer at a flow rate of 12 ml/min. After mixing, LNP suspension was immediately diluted in PBS (Corning, Manassas, USA). Then, the formulation was reconcentrated by centrifugation at 2000 g in Amicon filters (30,000 MWCO, Amicon Ultra-15 Centrifugal Filter Unit, Millipore Sigma, Burlington, USA). Finally, the LNP suspension was filtered using a 0.22 micron syringe filter and kept at 4°C until use. Free and total RNA concentration were determined by Ribogreen assay (Quant-iT RiboGreen RNA, Invitrogen, Carlsbad, USA). For obtaining total RNA concentration, LNPs were lysed for 30 min at 37°C in Triton X-100 1%. Encapsulation was calculated as (total RNA-Free RNA)/(total RNA x 100). Particle sizes were measured by Dynamic Light Scattering in a DynaPro NanoStar instrument (Wyatt Technology, Santa Barbara, USA) and analyzed with Dynamics 8.0 software (Wyatt Technology, Santa Barbara, USA). Samples were diluted in PBS (Corning, Manassas, USA) until full laser power could be used to record the signal. For each sample, 3 measurements were conducted, each consisting of 10 recordings of 10 s.
10
Quantifying Viral DNA Internalization
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Cells were plated into 24-well plates and the next day infected with virus at 1 or 10 pfu/cell in 200 µL of appropriate cold media. The cells were immediately incubated in a cold room (4 °C) for 2 h, allowing viruses to bind but preventing internalization of the virus into the cells. Cells were gently washed with PBS (Corning, New York, NY, USA) to remove unbound viruses. Then 200 µL of PBS (Corning, New York, NY, USA) with 20 µL of proteinase K was added to each well, and lysis was performed using lysis buffer from a QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany). Using the manufacturer recommendation, the same kit was used to extract DNA, which was eluted and quantified using a Nanodrop Lite spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). qPCR was then performed using E4 primers (Forward 5′-GGAGTGCGCCGAGACAAC-3′, Reverse 3′-ACTACGTCCGGCGTTCCAT-5′) and TaqMan (Probe 5′-G-FAM-TGGCATGACACTACGACCAACACGATCT-TAMRA-3′) on a LightCycler® 480 II (Roche, Basel, Switzerland). Copies of E4 were normalized to 50 ng of total DNA.
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