Human serum

Manufactured by Thermo Fisher Scientific

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Human serum is a biological fluid that is obtained from the blood of human individuals. It is composed of various proteins, electrolytes, and other biomolecules. The core function of human serum is to serve as a sample matrix for clinical and diagnostic laboratory testing.

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Human AB serum (119 citations) Human endothelial serum-free medium (22 citations) Normal human serum (5 citations) IMDM + 10% AB human serum (5 citations) Human endothelial cell serum-free media (4 citations) Human keratinocyte growth serum (4 citations) Heat-inactivated human AB serum (4 citations) Normal human AB serum (2 citations) Human Endothelial Serum-Free Media (2 citations) AIM-V + 10% human AB serum (2 citations)

89 protocols using human serum

Ex vivo Expansion of LAK Cells

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PBMCs from an adult healthy donor were expanded ex vivo in RPMI medium containing Interleukin-2 (IL-2) (ThermoFisher Scientific, cat. # PHC0023) plus 5% human serum (ThermoFisher Scientific, cat. # 34005–100, discontinued) for 20 days as described.^68,71 At the end of this expansion period, the LAK (Lymphokine Activated Killer) cell population consisted of approx. 25% NK cells, 70% T cells and 5% NKT cells. LAK cells were then frozen in aliquots for subsequent use. Prior to use in cytolysis experiments, the cells were thawed and cultured overnight in RPMI medium containing 5% human serum (Invitrogen) plus 50 units/ml and 50 µg/ml PS, respectively, but no additional IL-2.

Braciak T.A., Roskopf C.C., Wildenhain S., Fenn N.C., Schiller C.B., Schubert I.A., Jacob U., Honegger A., Krupka C., Subklewe M., Spiekermann K., Hopfner K.P., Fey G.H., Aigner M., Krause S., Mackensen A, & Oduncu F.S. (2018). Dual-targeting triplebody 33-16-123 (SPM-2) mediates effective redirected lysis of primary blasts from patients with a broad range of AML subtypes in combination with natural killer cells. Oncoimmunology, 7(9), e1472195.

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PARPi-FL Stability in Human Serum

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Human serum was purchased from Thermo Fisher Scientific. PARPi-FL NE was incubated in Human serum (25% nanoemulsion and 75% of serum to get a final volume of 1 mL) at 37°C over a period of 24 h. Fifty microliters of the NE−serum mixture was taken at different time points (1, 2, 4, 6, 12, and 24 h). The samples were analyzed on a Shimadzu HPLC equipped with a SPD-M20A UV detector, a LC-20AB pump system, a CBM-20A communication BUS module, and a RF-20A xs fluorescence detector (excitation = 498 nm, emission = 510 nm). SEC experiments were performed on a Superdex 10/300 column (GE Healthcare Life Sciences, Pittsburgh, PA) using PBS as eluent at a flow rate of 1 mL/min, where both absorbance and fluorescence were recorded.

Gonzales J., Kossatz S., Roberts S., Pirovano G., Brand C., Pérez-Medina C., Donabedian P., de la Cruz M.J., Mulder W.J, & Reiner T. (2018). Nanoemulsion-Based Delivery of Fluorescent PARP Inhibitors in Mouse Models of Small Cell Lung Cancer. Bioconjugate chemistry, 29(11), 3776-3782.

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Activation and Culture of Human T Cells

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Cells were cultured in RPMI-1640 medium supplemented with 2 mM glutamine, 1% (v/v) non-essential amino acids, 1% (v/v) sodium pyruvate, penicillin (50 U ml⁻¹), streptomycin (50 μg ml⁻¹; all from Invitrogen), and 5% (v/v) human serum (Swiss Blood Center, Basel). Human T cells were activated with plate-bound anti-CD3 (5 μg/ml, clone TR66) and anti-CD28 (1 μg/ml, clone CD28.2, BD Biosciences) for 48 h in 96-well Nunc Maxisorb plates. 1.5×10⁵ T cells were plated per well. After 48 h of activation, cells were transferred to 96-well U-bottom plates and cultured in IL-2 containing media (500 U/ml). In experiments, in which T cells that were cultured without a stimulus, 1.5×10⁵ T cells were plated in 96-well U-bottom plates and 200 μ1 of culture medium was added per well. Drugs were added at the following concentrations: CHX (50 μg/ml) and bortezomib/PS341 (10 μM).
For SILAC experiments, SILAC RPMI-1640 (GIBCO) was supplemented with 73 mg/l Lys-8 HCl and 42 mg/l Arg-10 HCl, 2 mM glutamine, 1% (v/v) sodium pyruvate, penicillin (50 U/ml), streptomycin (50 μg/ml; all from Invitrogen), and 5% (v/v) dialyzed human serum.

Wolf T., Jin W., Zoppi G., Vogel I.A., Akhmedov M., Bleck C.K., Beltraminelli T., Rieckmann J.C., Ramirez N.J., Benevento M., Notarbartolo S., Bumann D., Meissner F., Grimbacher B., Mann M., Lanzavecchia A., Sallusto F., Kwee I, & Geiger R. (2020). Dynamics in protein translation sustaining T cell preparedness. Nature immunology, 21(8), 927-937.

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HCV Infection Dynamics in Primary Human Hepatocytes

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PHHs were obtained from XenoTech LLC, Kansas City, MO, and cultured with hepatocyte culture media supplemented with 10% human serum (Invitrogen, Brown Deer, WI). After 24 hours they were infected with cell culture grown HCV-GFP chimera virus with a MOI (multiplicity of infection) of 0.1 described earlier^{42 (link)}. After 18 hours of infection, hepatocytes were replaced with fresh hepatocyte culture media (XenoTech, LLC, Kansas City, MO) supplemented with 10% human serum (Invitrogen, Brown Deer, WI). Uninfected or infected PHHs were harvested every 3 days and cell pellets were used for RNA isolation and Western blot analysis. The success of HCV replication in the infected PHHs was assessed by the detection of positive strand HCV RNA levels by reverse transcription real-time quantitative PCR (QRT-PCR) and Western blot analysis of HCV NS3 protein.

Aydin Y., Chedid M., Chava S., Danielle Williams D., Liu S., Hagedorn C.H., Sumitran-Holgersson S., Reiss K., Moroz K., Lu H., Balart L.A, & Dash S. (2017). Activation of PERK-Nrf2 oncogenic signaling promotes Mdm2-mediated Rb degradation in persistently infected HCV culture. Scientific Reports, 7, 9223.

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Hepatocyte Culture and HCV Infection

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PHHs were obtained from XenoTech, LLC (Kansas City, MO) and cultured with hepatocyte culture media supplemented with 10% human serum (Invitrogen, Brown Deer, WI). After 24 hours, they were infected with cell culture‐grown HCV (JFH‐ΔV3‐Rluc virus, HCV genotype 2a) with a multiplicity of infection (MOI) of 0.1, using a standard protocol of our laboratory. After 18 hours of infection, hepatocytes were replaced with fresh hepatocyte culture media (XenoTech) supplemented with 10% human serum (Invitrogen). Uninfected or infected PHHs were harvested every 3 days, and cell pellets were used for RNA isolation and western blot analysis. The success of HCV replication in the infected PHHs was assessed by the detection of positive‐strand HCV RNA levels by reverse transcription (RT) real‐time quantitative polymerase chain reaction (PCR) and western blot analysis of HCV NS3 protein.

Aydin Y., Chatterjee A., Chandra P.K., Chava S., Chen W., Tandon A., Dash A., Chedid M., Moehlen M.W., Regenstein F., Balart L.A., Cohen A., Lu H., Wu T, & Dash S. (2017). Interferon‐alpha‐induced hepatitis C virus clearance restores p53 tumor suppressor more than direct‐acting antivirals. Hepatology Communications, 1(3), 256-269.

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Activation and Culture of Human T Cells

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PBMC Expansion with HRV Peptides

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Peripheral blood mononuclear cells (PBMCs) were isolated from donor's blood samples (30 mL) by a density gradient on FicollPaque PLUS (Amershan). For in vitro expansions, PBMCs were cultured in RPMI 1640 (Gibco, NY, USA) supplemented with 10% human serum (Gibco NY, USA), 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM l‐glutamine (Lonza, Walkersville, USA) at a density of 2 × 10⁶ cells/mL in 24‐well plates (BD Biosciences) and stimulated with individual HRV peptides (10 µM final concentration) and IL‐2 (10 U/mL; Immunotools). Cells were kept at 37°C in 5% CO2 for 6 days being split and fed as necessary with an additional doses of IL‐2 (10 U/mL; Immunotools) and individual HRV peptides (10 µM) at day 3 of culture. Expanded cells were washed twice with PBS (Gibco, NY, USA) and let rest for 4 hours in RPMI 1640 without both, human serum and stimulation, prior to any functional assay.

Gomez‐Perosanz M., Sanchez‐Trincado J.L., Fernandez‐Arquero M., Sidney J., Sette A., Lafuente E.M, & Reche P.A. (2020). Human rhinovirus‐specific CD8 T cell responses target conserved and unusual epitopes. The FASEB Journal, 35(1), e21208.

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Hepatitis B Virus Immune Response Modulation

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PMBCs were thawed and 1 × 10⁶ cells/mL were cultured at 37 °C in a 5% CO₂ incubator for 10 d in AIM V (Gibco, USA) supplemented with 5% human serum (Thermo Fisher Scientific, USA) in round-bottomed 96-well plates. Cells were stimulated with 1 μg/mL overlapping peptides of HBV core (PM-HBV-CPULTRA) or HBV envelope (env) (PM-HBV-LEPULTRA) from JPT in the presence or absence of the different compounds tested. These compounds included polyphenols, resveratrol 5 μM and oleuropein 0.25 μM (APExBIO, USA); iACAT, Avasimibe 0.25 μM (Selleck, USA) and K-604 0.05 μM (MCE, USA); and MTA, Mito-TEMPO 5 μM (Merk, Germany) and MitoQ 0.05 μM (MCE). PBMCs were cultured for 10 d in the presence of recombinant IL-2 (20 IU/mL), IL-7 (10 ng/mL), and IL-15 (10 ng/mL) (Biolegend, USA).

Fu Y.L., Zhou S.N., Hu W., Li J., Zhou M.J., Li X.Y., Wang Y.Y., Zhang P., Chen S.Y., Fan X., Song J.W., Jiao Y.M., Xu R., Zhang J.Y., Zhen C., Zhou C.B., Yuan J.H., Shi M., Wang F.S, & Zhang C. (2023). Metabolic interventions improve HBV envelope-specific T-cell responses in patients with chronic hepatitis B. Hepatology International, 17(5), 1125-1138.

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Conjunctival Tissue Culture Protocol

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To define the impact of extracellular matrix hydrogels on gel-forming mucin secretion, we used a previously published method to obtain a co-cultivation of stratified squamous cells, goblet cells, and undifferentiated/stem cells [56 (link)]; starting with mincing the isolated conjunctival tissue to maintaining the cultures as described in 4.2.2. The medium was composed of Dulbecco’s Modified Eagle Medium (DMEM)/F12 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 100 µg/mL penicillin/streptomycin (Lonza, Basel, Switzerland), 1 µg/mL insulin (Sigma-Aldrich, St. Louis, MO, USA), 0.5 µg/mL hydrocortisone (Sigma-Aldrich, St. Louis, MO, USA), 2 ng/mL rat EGF (PeproTech, Cranbury, NJ, USA), and 10% human serum (Thermo Fisher Scientific, Waltham, MA, USA). The explant conjunctival cultures were photographed using an EVOS microscope (Thermo Fisher Scientific, Waltham, MA, USA).

Van Acker S.I., Van den Bogerd B., Van Acker Z.P., Vailionytė A., Haagdorens M., Koppen C., Ní Dhubhghaill S., Dartt D.A, & Pintelon I. (2021). Characterisation of Gel-Forming Mucins Produced In Vivo and In Ex Vivo Conjunctival Explant Cultures. International Journal of Molecular Sciences, 22(19), 10528.

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Comprehensive Cell Culture Protocol

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RPMI, DMEM/F12 media, phosphate-buffered saline (PBS), HEPES, sodium pyruvate, glutamine and penicillin/streptomycin were purchased from Lonza (Portsmouth, New Hampshire, USA). Fetal bovine serum was from Atlanta Biologicals (Flowering Branch, Georgia, USA). Human serum, human insulin, Alamar Blue, calcein AM/ethidium homodimer-1 (EH-1) live/dead assay kit, antibodies against cytokeratin 4 (CK4), cytokeratin 7 (CK7), anti-Ki-67 antibody and vimentin were provided by ThermoFisher (Waltham, Massachusetts, USA). Additional CK4 and CK7 antibodies were purchased from SantaCruz Biotechnology (Dallas, Texas, USA). PI solution (10%) was obtained from CVS (Woonsocket, Rhode Island, USA). Hydrogen peroxide, hydrocortisone, epidermal growth factor (EGF), fluorescein isothiocyanate (FITC)-conjugated lectin from Ulex europaeus agglutinin I (UEA) and lectin Bandeiraea Simplicifolia agglutinin conjugated to FITC were provided by Sigma-Aldrich (St Louis, Missouri, USA). MUC5AC antibody was purchased from Abcam (Cambridge, Massachusetts, USA). Secondary antibodies conjugated to Cy 2 or Cy 3 were purchased from Jackson ImmunoResearch Laboratories (West Grove, Pennsylvania).

Swift W., Bair J.A., Chen W., Li M., Lie S., Li D., Yang M., Shatos M.A., Hodges R.R., Kolko M., Utheim T.P., Scott W, & Dartt D.A. (2020). Povidone iodine treatment is deleterious to human ocular surface conjunctival cells in culture. BMJ Open Ophthalmology, 5(1), e000545.

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