Blood samples from diagnosed PNH patients (Table 1 ) were collected with consent following a Cleveland Clinic IRB approved protocol. WT mice and CD55/CD59 double knockout mice(17 (link)) were maintained under pathogen-free conditions at Cleveland Clinic. All the procedures involving the animals were approved by the Institutional Animal Care and Use Committee of Cleveland Clinic. Pooled normal human serum (NHS) was purchased from Innovative Research Inc (Novi, MI).
Pooled normal human serum
Manufactured by Innovative Research
Sourced in United States
Pooled normal human serum is a biological material composed of the liquid component of blood, obtained from a pool of healthy human donors. It contains a variety of proteins, electrolytes, and other substances naturally present in human blood serum.
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Lab products found in correlation
Multiskan fc, by Thermo Fisher Scientific (2 mentions)
Incucyte, by Sartorius (1 mentions)
Propidium iodide, by BD (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
96 well plate, by Corning (1 mentions)
Cytexpert software, by Beckman Coulter (1 mentions)
Wild type c57bl 6 mice, by Jackson ImmunoResearch (1 mentions)
F0895, by Merck Group (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Cholesterol chol, by Merck Group (1 mentions)
Celltiter glo 3d cell viability assay reagent, by Promega (1 mentions)
Propidium iodide flow cytometry kit, by Abcam (1 mentions)
Celltiter glo, by Promega (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
1200 series reverse phase hplc, by Agilent Technologies (1 mentions)
Rotor gene, by Qiagen (1 mentions)
Pooled human serum, by Innovative Research (1 mentions)
Nuclisens easymag, by bioMérieux (1 mentions)
10 protocols using pooled normal human serum
1
PNH Patient Blood Collection and Mice Study
2
PNH Patient Blood Collection and Mice Study
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Blood samples from diagnosed PNH patients (Table 1 ) were collected with consent following a Cleveland Clinic IRB approved protocol. WT mice and CD55/CD59 double knockout mice(17 (link)) were maintained under pathogen-free conditions at Cleveland Clinic. All the procedures involving the animals were approved by the Institutional Animal Care and Use Committee of Cleveland Clinic. Pooled normal human serum (NHS) was purchased from Innovative Research Inc (Novi, MI).
3
Flow Cytometry and IncuCyte Cell Viability Assays
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Cells cultured in 24-well plates (2 cm diameter) were treated as indicated in the figure legends. After treatment, both media and trypsinized adherent cells were harvested and stained for propidium iodide (BD Bioscience) as described by the manufacturer. Samples were then processed for by flow cytometry on the CytoFLEX (Beckman Coulter, Brea, CA, USA) and 10,000 independent events were analyzed using CytExpert software (Beckman Coulter).
Cell viability assays were also performed using the IncuCyte instrument (Sartorius) as previously described [30 (link)]. Briefly, uninfected (mock) or OC43 infected H1299-NLR cells were plated in triplicate in 96-well plates (Corning) at 7000 cells/well. Plates were maintained in the IncuCyte CO2 incubator for 3–4 days (d), while images were captured every 2 h using 10× objective in red and phase channels. Red object count (ROC) per well was calculated and scans were normalized to the value at time zero (ROCt0) when treatments were added. Pooled normal human serum (NHS) was purchased (Innovative Research, Novi, MI, USA). Sera were heat inactivated (HI) by heating to 56 °C for 30 min. C3, C5, C6, C7-depleted sera and their respective purified recombinant proteins were purchased from Complement Technologies (Tyler, TX, USA).
Cell viability assays were also performed using the IncuCyte instrument (Sartorius) as previously described [30 (link)]. Briefly, uninfected (mock) or OC43 infected H1299-NLR cells were plated in triplicate in 96-well plates (Corning) at 7000 cells/well. Plates were maintained in the IncuCyte CO2 incubator for 3–4 days (d), while images were captured every 2 h using 10× objective in red and phase channels. Red object count (ROC) per well was calculated and scans were normalized to the value at time zero (ROCt0) when treatments were added. Pooled normal human serum (NHS) was purchased (Innovative Research, Novi, MI, USA). Sera were heat inactivated (HI) by heating to 56 °C for 30 min. C3, C5, C6, C7-depleted sera and their respective purified recombinant proteins were purchased from Complement Technologies (Tyler, TX, USA).
4
Recombinant SSL7 Protein Purification
5
Quantifying Plasma Protein Adsorption
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Bradford assay was used to study the time-course of blood plasma proteins and of pure FBN adsorption to chitosan with or without aptamers.
To this purpose, discs were soaked for 1 h in 200 µL of PBS supplemented with 2% of human serum (Pooled Normal Human Serum, Innovative Research, Peary Court, FL, USA) either of pure FBN (F0895, Sigma-Aldrich) at a concentration comparable with that present in a 2% human serum solution (0.02 mg/mL). Protein amount in supernatants was quantitated after 5, 15, 30, 45 and 60 min through Bradford assay (BIO-RAD Protein Assay, BIO-RAD, Hercules, CA, USA) according to the manufacturer’s recommendations. Ten-µl aliquots were collected at each time point, mixed with 200 µL of Bradford Working Solution and incubated at 37 °C for 2 min prior to absorbance assessment at 620 nm with a micro plate photometer (Multiskan™ FC, Thermo Fisher Scientific, Waltham, MA, USA). The number of proteins deposited on the discs was finally calculated by subtracting the residual concentration in supernatant from the initial one.
To this purpose, discs were soaked for 1 h in 200 µL of PBS supplemented with 2% of human serum (Pooled Normal Human Serum, Innovative Research, Peary Court, FL, USA) either of pure FBN (F0895, Sigma-Aldrich) at a concentration comparable with that present in a 2% human serum solution (0.02 mg/mL). Protein amount in supernatants was quantitated after 5, 15, 30, 45 and 60 min through Bradford assay (BIO-RAD Protein Assay, BIO-RAD, Hercules, CA, USA) according to the manufacturer’s recommendations. Ten-µl aliquots were collected at each time point, mixed with 200 µL of Bradford Working Solution and incubated at 37 °C for 2 min prior to absorbance assessment at 620 nm with a micro plate photometer (Multiskan™ FC, Thermo Fisher Scientific, Waltham, MA, USA). The number of proteins deposited on the discs was finally calculated by subtracting the residual concentration in supernatant from the initial one.
6
Formulation and Characterization of Dox-Loaded Lipid Nanoparticles
7
Complement Activation Assay Protocols
8
Serum Stability of MARCKS Peptides
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The serum stability of L- and D-MARCKS ED was measured as in a previously described protocol51 (link). Briefly, 100 μg ml−1 L- or D-MARCKS ED was incubated at 37 °C in RPMI 1640 medium (Fisher Scientific, Pittsburgh, PA) supplemented with 25% (v/v) pooled normal human serum (Innovative Research, Novi, MI). At specific time points, the reaction was subsampled and a trichloroacetic acid solution was added to a final concentration of 5% (w/v). Each subsample mixture was cooled for 15 min at 4 °C, then centrifuged at 16,000 g for 4 min to precipitate the serum proteins. The resulting supernatant was analyzed with a 1200 Series reverse phase HPLC (Agilent Technologies) using a semi-prep C18 column. Non-degraded peptide was quantified by setting the UV-visible HPLC detection to 480 nm and integrating the chromatogram peaks with retention times matching that of the whole peptide (39 to 40 min). Peaks at 480 nm are due to the absorbance NBD fluorophore label. Subsamples from serum mixtures without added MARCKS ED showed no peaks in this retention time range.
9
Limit of Detection for HAV RT-PCR
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Pooled normal human serum (Innovative Research) was spiked with pUC57-5′-UTR-HAV and a ten-fold dilution series from 1011 to 103 copies/mL was produced. Two hundred microliters of each dilution as well as negative serum control samples were extracted in triplicate using the NucliSENS easyMag (bioMérieux, Craponne, France). The RT-PCR was performed on the Qiagen Rotor-Gene using primers HAVF1 and HAVR2 to approximate a concentration range for determining the limit of detection starting at the lowest concentration at which the plasmid was detected in 100% of the triplicate samples. Based on the RT-PCR data from the ten-fold dilution series, pUC57-5′-UTR-HAV was spiked into pooled human serum (Innovative Research, Novi, MI, USA) and a five-fold dilution series from 104 to 80 copies/mL was produced. Ten replicates of 200 μL of each dilution and three replicates of negative serum samples were extracted using the NucliSENS easyMag. The RT-PCR was performed as previously described to determine the lowest concentration at which the plasmid was detected in 100% of the ten replicates.
10
RSV Neutralization Assay with Serum Controls
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Pooled normal human serum (Innovative Research, Novi, MI) with a mean microneutralization titer of 9.72 log2 IC50 was used as a positive control (PC) on each sample plate to evaluate assay performance. Prior to use in the assay, the pooled serum was heat inactivated at 56°C for 45 min, aliquoted, and stored at −80°C for individual assay use. Monoclonal antibodies were purchased from EMD Millipore (Billerica, MA) or were acquired internally. Human sera with RSV-neutralizing activity from natural RSV infections were purchased from Bioreclamation IVT (Hicksville, NY). Cynomolgus macaque sera were obtained from preclinical studies of RSV vaccine candidates (10 (link)). All samples were heat inactivated at 56°C for 45 to 60 min to remove complement activity and then stored at −80°C prior to testing.
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