Normal human serum purchased from Sigma-Aldrich Chemical Company was used and its specifications are listed in Table S1.† Extraction with a chloroform–methanol mixture was based on the methodology of Folch et al.36 After 4 mL of Normal human serum was added to 10 mL of chloroform–methanol (2 : 1, v/v), the mixture was agitated manually for 1 minute and centrifuged at 4000 rpm for 10 minutes at rt. After centrifugation, the aqueous phase was collected and repeated once more with hexane.37 (link)
Normal human serum
Manufactured by Merck Group
Sourced in United States, Germany, Sao Tome and Principe
Normal human serum is a laboratory product that serves as a reference material for various clinical and diagnostic tests. It is a pooled, sterile-filtered serum derived from healthy adult human donors. The product provides a consistent and well-characterized source of human serum components, including proteins, enzymes, and other biomolecules, for use in quality control, method validation, and calibration procedures.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Thermo Fisher Scientific (4 mentions)
Protein assay dye reagent concentrate, by Bio-Rad (3 mentions)
Albumin determination reagent bromocreosol purple, by Merck Group (2 mentions)
Mixed mode solid phase extraction cartridges, by Waters Corporation (2 mentions)
Ammonium hydroxide, by Merck Group (2 mentions)
Formic acid, by Merck Group (2 mentions)
Boric acid, by Merck Group (2 mentions)
Macs ls column, by Miltenyi Biotec (2 mentions)
Rpmi 1640, by Merck Group (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Facscanto 2, by BD (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Tca solution, by Merck Group (2 mentions)
O phthaldialdehyde opa, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Reversed phase sep pak c18 cartridges, by Waters Corporation (1 mentions)
Ammonium acetate, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
42 protocols using normal human serum
1
Lipid Extraction from Human Serum
2
Aflatoxin B1-Lysine Adduct Quantification
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AFB1-lysine adduct standard was synthesized and purified as previously described by Sabbioni et al. [39 (link)]. Albumin determination reagent (bromocreosol purple), and normal human serum were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO). Pronase (25 kU, Nuclease-free) was purchased from Calbiochem (La Jolla, CA). Protein assay dye reagent concentrate and protein standards were purchased from Bio-Rad Laboratories Inc. (Hercules, CA). Boric acid, o-phthaldialdehyde (OPA), 2-mercaptoethanol, FB1 from F. verticilioides (~ 98% purity, TLC), 10× phosphate buffered saline (PBS), ammonium hydroxide, ammonium acetate, sodium chloride, sodium phosphate monobasic, hydrochloric acid, and formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). OPA reagents were prepared by dissolving 10 mg of OPA and 30 μl of 2-mercaptoethanol in 250 μl of methanol and mixing with 4.75 ml of 3% Boric acid buffer (pH 10.5) and stored at 4 °C avoiding light before use. Mixed mode solid phase extraction (SPE) cartridges, as well as Sep-Pak reversed phase C18 cartridges were purchased from the Waters Corp. (Milford, MA). All other chemicals and solvents were of highest grade and purity available.
3
Cryopreservation and Thawing of PBMCs
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Healthy donor (Australian Red Cross Blood Services) PBMCs were isolated by Ficoll (Amersham Pharmacia Biotech, Sweden) density gradient centrifugation. The isolated PBMCs were suspended in cryovials containing a freeze medium mixture of 10% DMSO (Sigma-Aldrich, USA) and 90% heat-inactivated fetal calf serum (GIBCO, Life Technologies, USA) and frozen at a speed of −1°C/min in a −80°C freezer then subsequently stored in liquid nitrogen. Prior to cell culture, each vial of frozen PBMCs was rapidly thawed in a 37°C water-bath and resuspended in complete AIM V media [AIM V (Life Technologies, USA) supplemented with 5% normal human serum (Sigma-Aldrich, USA)].
4
Radiochemical Characterization of 64Cu-Pembrolizumab Tracer
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Radio-thin layer paper chromatography (R-TLC) and SEC. 2000 radio-HPLC were performed to assess the tracer quality. Immunoreactivity and serum stability were carried out as per published methods described elsewhere30 (link),31 (link). The 64Cu-pembrolizumab tracer serum stability was assayed with normal human serum (Sigma Aldrich, St Louis, MO) and incubated at 37 °C. Briefly, tracer and serum were mixed for 6 h. After 6 h, 100 μL was aliquoted and analyzed by radio-HPLC and the eluent was collected in 1-mL fractions and counted in a gamma counter. Percent activity of each fraction was computed to measure tracer stability in serum. Tracer pyrogenicity and sterility tests were performed as per previously published procedures32 .
5
Evaluating Complement Activation by AuNPs
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AuNPs (2 μg
mL–1, 100 μL) were incubated in commercially
available normal human serum (100 μL) (Sigma-Aldrich, St. Louis,
MO) for 1 h at 37 °C. PBS (10 mM, 100 μL) was used as the
negative control. The mixture was then centrifuged to isolate AuNPs,
and the serum-containing supernatant (100 μL) was used to analyze
the concentration of the final product of complement activation, SC5b-9,
induced by AuNPs of different configurations using an ELISA kit, following
the procedure provided by the kit (Human TCC C5b-9, Biosite).
mL–1, 100 μL) were incubated in commercially
available normal human serum (100 μL) (Sigma-Aldrich, St. Louis,
MO) for 1 h at 37 °C. PBS (10 mM, 100 μL) was used as the
negative control. The mixture was then centrifuged to isolate AuNPs,
and the serum-containing supernatant (100 μL) was used to analyze
the concentration of the final product of complement activation, SC5b-9,
induced by AuNPs of different configurations using an ELISA kit, following
the procedure provided by the kit (Human TCC C5b-9, Biosite).
6
Aflatoxin B1 Detection in Serum
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AFB1 (> 98% purity), albumin determination reagent bromocreosol purple, and normal human serum were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA). Pronase (25 kU, Nuclease‐free) was purchased from Calbiochem (La Jolla, CA, USA). Protein assay dye reagent concentrate and protein standards were purchased from Bio‐Rad Laboratories Inc. (Hercules, CA, USA). Mixed mode solid phase extraction cartridges were purchased from the Waters Corp. (Milford, MA, USA). Authentic AFB‐Lys was synthesized as previously described (Sabbioni, Skipper, Büchi, & Tannenbaum, 1987 ). All other chemicals and solvents used were of highest grade commercially available.
7
Flow cytometry analysis of macrophages
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Cells were harvested by scraping and resuspended in FACS staining buffer (0.9% normal mouse serum, 0.9% normal rabbit serum, 0.9% normal human serum [all Sigma-Aldrich], 3% BSA, 2 mM EDTA in PBS). After antibody staining and washing, cells were resuspended in PBS with Hoechst (BD Biosciences). Counting was performed using a FACSCanto-II (BD Biosciences), and data were analyzed with FlowJo software (TreeStar) by gating on F4/80 and CD11b positive cells.
8
Serum Bactericidal Assay for K. pneumoniae
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Twenty‐five microlitres of 2.5 × 105 CFU of K. pneumoniae in 1× PBS (Thermo Scientific) were inoculated into wells of a 96‐well plate containing 75 μl of 100% pooled normal human serum (Sigma‐Aldrich). Control serum was inactivated by heating at 56°C for 30 min prior to incubation of bacteria. The plate was then incubated at 37°C. At 0, 1, 2 and 3 hr post inoculation, appropriate dilutions were plated on LB agar to enumerate surviving bacterial colony‐forming units (CFU).
9
Bactericidal Assay of H. influenzae
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Overnight growth of enriched H. influenzae biogroup aegyptius ON-OFF strain pairs was harvested from sBHI plates and was used to inoculate sBHI broth and incubated at 37°C for 4–6 hours. Broth cultures were then harvested via centrifugation, washed once with Hank’s balanced salt solution (HBSS), and resuspended to an A600 of 0.2. Ten microliters of the bacterial suspension (~2.0 × 104 cfu) was added to a final volume of HBSS containing 16.6% normal human serum (Sigma-Aldrich) and 10% rabbit complement (Sigma-Aldrich). HBSS only, serum only, and complement only were included as controls. HBSS assays were incubated at 37°C for 1 hour prior to enumeration on supplemented BHI plates. All input cultures were also enumerated prior to incubation, and survival was calculated as a percentage of the corresponding input cfu.
10
HIV-1 Binding Antibody Multiplex Assay
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