Proteins, fluorescamine, SYPRO Orange, urea, ammonium bicarbonate, 1,4-dithiothreitol (DTT), iodoacetamide (IAM), formic acid (FA), trypan blue, and pooled human serum were purchased from Sigma-Aldrich (St. Louis, MO). Ultrapure water with electric resistance > 18.2 MΩ was produced in-house by the Millipore Milli-Q water purification system (Billerica, MA).
Pooled human serum
Manufactured by Merck Group
Sourced in Germany, United States
Pooled human serum is a laboratory reagent composed of pooled serum from multiple healthy human donors. It is used as a reference material for various analytical and testing applications in the clinical laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (4 mentions)
Lymphoprep, by STEMCELL (4 mentions)
Macrophage colony stimulating factor, by R&D Systems (3 mentions)
Human serum type ab, by Merck Group (2 mentions)
Roswell park memorial institute (rpmi), by Merck Group (2 mentions)
Urea, by Merck Group (2 mentions)
Nafion 117, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Sequencing grade trypsin, by Promega (2 mentions)
Dithiothreitol (dtt), by Merck Group (2 mentions)
Lc ms grade acetonitrile, by Honeywell (2 mentions)
Sypro orange, by Merck Group (1 mentions)
1 4 dithiothreitol, by Merck Group (1 mentions)
Milli q water purification system, by Merck Group (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Ammonium bicarbonate, by Merck Group (1 mentions)
Fluorescamine, by Merck Group (1 mentions)
Poly 1 c, by InvivoGen (1 mentions)
Cpg odn 2006, by InvivoGen (1 mentions)
16 protocols using pooled human serum
1
Protein Quantification and Sample Preparation
2
Isolation and Differentiation of Primary Human Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary MDM were prepared from fresh blood from healthy volunteers. The study was approved by the joint University College London/University College London Hospitals NHS Trust Human Research Ethics Committee and written informed consent was obtained from all participants.
Peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation using Lymphoprep (Stemcell Technologies), washed three times (PBS) and plated to select for adherent cells. Non-adherent cells were washed away after 1.5 h and the remaining cells incubated in RPMI (Gibco) supplemented with 10% heat-inactivated pooled human serum (Sigma) and 100 ng ml−1 macrophage colony stimulating factor (R&D systems). For replication experiments with full-length viruses, the medium was then refreshed after 3 d (RPMI 1640 with 10% human serum), removing any remaining non-adherent cells. After 6 d, media were replenished with RPMI containing 5% type AB human serum (Sigma-Aldrich). For single-round experiments with VSV-G-pseudotyped viruses, cells were washed (PBS) on day 3 of differentiation and the medium changed to RPMI supplemented with 10% heat-inactivated FBS. MDM were then infected 3–4 d later. Replicate experiments were performed with cells derived from different donors.
Peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation using Lymphoprep (Stemcell Technologies), washed three times (PBS) and plated to select for adherent cells. Non-adherent cells were washed away after 1.5 h and the remaining cells incubated in RPMI (Gibco) supplemented with 10% heat-inactivated pooled human serum (Sigma) and 100 ng ml−1 macrophage colony stimulating factor (R&D systems). For replication experiments with full-length viruses, the medium was then refreshed after 3 d (RPMI 1640 with 10% human serum), removing any remaining non-adherent cells. After 6 d, media were replenished with RPMI containing 5% type AB human serum (Sigma-Aldrich). For single-round experiments with VSV-G-pseudotyped viruses, cells were washed (PBS) on day 3 of differentiation and the medium changed to RPMI supplemented with 10% heat-inactivated FBS. MDM were then infected 3–4 d later. Replicate experiments were performed with cells derived from different donors.
3
Serum-based Immune Response Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pooled human serum from healthy donors was purchased from Sigma Aldrich. CpG ODN 2006, Poly I:C, LPS, and R848 were purchased from InvivoGen. ELISA kits were purchased as follows: HMGB-1 (Tecan- ST51011), Cell-Death Detection ELISA Plus (Roche-11,774,425,001), and albumin (Abcam-ab179887). Picogreen and Ribogreen stains were obtained from Life Technologies (P7589 and R41190). PAMAM-G3 based nucleic acid binding fiber and polyethylenimine (PEI)-based nucleic acid binding fiber were graciously provided by Jaewoo Lee, PhD and synthesized as previously described [15 ].
4
Isolation and Culture of Primary MDMs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary monocyte‐derived macrophages (MDMs) were prepared from fresh blood from healthy volunteers. The study was approved by the joint University College London/University College London Hospitals NHS Trust Human Research Ethics Committee, and written informed consent was obtained from all participants. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Lymphoprep (Stemcell Technologies). PBMCs were washed three times with PBS and plated to select for adherent cells. Non‐adherent cells were washed away after 1.5 h and the remaining cells incubated in RPMI (Gibco) supplemented with 10% heat‐inactivated pooled human serum (Sigma) and 40 ng/ml macrophage colony‐stimulating factor (R&D systems). Cells were further washed after 3 days and the medium changed to RPMI supplemented with 10% heat‐inactivated FBS. MDM was then infected 3–4 days later. Replicate experiments were performed with cells derived from different donors.
5
Generation of Cytokine-Matured Dendritic Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The monocytes were isolated from the PBMCs by plastic adherence. A total of 3–4 × 107 PBMCs per 10 cm tissue culture dish (Falcon, Corning GmbH, Kaiserslautern, Germany) were incubated in 10 ml DC medium for 1 h at 37°C. DC medium consisted of Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 1% heat-inactivated non-autologous human plasma from individual donors (Transfusionsmedizin, Universitätsklinikum Erlangen, Erlangen, Germany) or heat-inactivated pooled human serum (Sigma-Aldrich), 2 mM L-glutamine (Lonza), and 20 mg/l gentamycin (Lonza). The non-adherent fraction (NAF) was removed by rinsing with RPMI 1640 and used for T-cell isolation. Approximately 1–2 × 107 non-adherent cells were obtained from each dish. The adherent monocytes were differentiated to immature DCs over 6–7 d in DC medium supplemented with 800 IU/ml granulocyte macrophage colony-stimulating factor (GM-CSF) (Miltenyi, Bergisch Gladbach, Germany), and 250 IU/ml IL-4 (Miltenyi) on days 1, 3, and 5. On day 6 or 7 the DCs were matured by adding a cytokine cocktail of 200 IU/ml IL-1β (CellGenix, Freiburg, Germany), 1000 IU/ml IL-6 (CellGenix), 200 U/ml tumor necrosis factor (TNF) (Peprotech, Hamburg, Germany), and 1 μg/ml prostaglandin E2 (PGE2) (Pfizer, Zurich, Switzerland) for another 24 h. One culture dish usually yielded between 1–2.5 × 106 cytokine-matured DCs (cmDCs).
6
Cytotoxic Response of Anti-CD20 mAbs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cytotoxic drug response of three anti-CD20 mAbs, rituximab, ofatumumab, and obinutuzumab, was compared among 680 lines. LCLs were plated into 384-well plates at approximately 4000 cells/well in a total volume of 50 μL under both normal cell culture conditions (ofatumumab and obinutuzumab) or in the presence of 25% human serum (as a source of complement) and with or without 10 μg/mL of each antibody. In initial screening experiments, which were part of a larger drug screening project [23 (link)], pooled human serum (Sigma Aldrich) was used for 72 h, followed by overnight exposure to Alamar Blue (Invitrogen, Waltham, MA, USA) to assess viability. An Infinite F200 microplate reader with Connect Stacker (Tecan US, Chapel Hill, NC, USA) and iControl software (Version 1.6) was used to measure the fluorescence intensity of the viability dye at EX535nm and EM595nm. Viability was expressed relative to samples without antibodies using 10% DMSO samples for background subtraction. Each cell line was assayed twice as quadruplicates. In subsequent experiments, cells were treated for 24 h in the presence of pooled human serum (Innovative Research, Novi, MI, USA), followed by overnight incubation with Alamar Blue.
7
Activation and Isolation of CD4+ T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the unactivated (resting) CD4+ T cell samples, 1 × 107 CD4+ T cells from both donors were washed three times with PBS before the pellet was shock-frozen in liquid nitrogen and stored at −80 °C. For the activated CD4+ T cell samples, 1 × 107 CD4+ T cells from both donors were activated in anti-CD3 coated plates (clone OKT3, eBioscience, 5 µg/mL, 1 h) in the presence of 1 μg/mL anti-CD28 (clone CD28.2, eBioscience). CD4+ T cells were cultured for 72 h in RPMI 1640 (Sigma-Aldrich, Darmstadt, Germany), supplemented with 10% pooled human serum (The Blood Bank, St Olav’s Hospital, Trondheim, Norway) at 37 °C and 5% CO2. CD4+ T cells were washed three times with PBS before the pellet was shock-frozen in liquid nitrogen and stored at −80 °C.
8
Isolation and Cultivation of Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary monocyte‐derived macrophages (MDM) were prepared from fresh blood from healthy volunteers. The study was approved by the joint University College London/University College London Hospitals NHS Trust Human Research Ethics Committee, and written informed consent was obtained from all participants. Experiments conformed to the principals set out in WMA declaration of Helsinki and the Department of Health and Human Services Belmont Report. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Lymphoprep (Stemcell Technologies). PBMCs were washed three times with PBS and plated to select for adherent cells. Non‐adherent cells were washed away after 2 h and the remaining cells incubated in RPMI (Gibco) supplemented with 10% heat‐inactivated pooled human serum (Sigma) and 100 ng/ml macrophage colony‐stimulating factor (PeproTech). The medium was replaced after 3 days with RPMI with 5% FCS, removing any remaining non‐adherent cells. Cells were infected or treated with conditioned media 3–4 days later.
9
Primary monocyte-derived macrophage isolation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary monocyte-derived macrophages (MDM) were prepared from fresh blood from healthy volunteers. The study was approved by the joint University College London/University College London Hospitals NHS Trust Human Research Ethics Committee and written informed consent was obtained from all participants.
Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Lymphoprep (Stemcell Technologies). PBMCs were washed three times with PBS and plated to select for adherent cells. Nonadherent cells were washed away after 1.5 h and the remaining cells incubated in RPMI (Gibco) supplemented with 10 % heat-inactivated pooled human serum (Sigma) and 100 ng/ml macrophage colony stimulating factor (R&D systems). For replication experiments with full-length viruses, the medium was then refreshed after 3 days (RPMI 1640 with 10% human serum), removing any remaining non-adherent cells. After 6 days, media was replenished with RPMI containing 5% type AB human serum (Sigma-Aldrich). For single-round experiments with VSV-G-pseudotyped viruses, on day 3 of differentiation cells were further washed and the medium changed to RPMI supplemented with 10 % heat-inactivated FBS. MDM were then infected 3-4 days later. Replicate experiments were performed with cells derived from different donors.
Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Lymphoprep (Stemcell Technologies). PBMCs were washed three times with PBS and plated to select for adherent cells. Nonadherent cells were washed away after 1.5 h and the remaining cells incubated in RPMI (Gibco) supplemented with 10 % heat-inactivated pooled human serum (Sigma) and 100 ng/ml macrophage colony stimulating factor (R&D systems). For replication experiments with full-length viruses, the medium was then refreshed after 3 days (RPMI 1640 with 10% human serum), removing any remaining non-adherent cells. After 6 days, media was replenished with RPMI containing 5% type AB human serum (Sigma-Aldrich). For single-round experiments with VSV-G-pseudotyped viruses, on day 3 of differentiation cells were further washed and the medium changed to RPMI supplemented with 10 % heat-inactivated FBS. MDM were then infected 3-4 days later. Replicate experiments were performed with cells derived from different donors.
10
Quantitative Analysis of Tofacitinib
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tofacitinib citrate and deuterium-labelled tofacitinib (tofacitinib-d3) were purchased from Cayman Chemical (Michigan, USA). LC-MS grade acetonitrile (ACN), water, pooled human serum and formic acid were purchased from Merck (Dorset, United Kingdom).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!