Human ab serum

Manufactured by Thermo Fisher Scientific

Sourced in United States, France, Germany, Australia

Human AB serum is a cell culture medium supplement derived from the blood of individuals with the AB blood type. It provides a source of proteins, growth factors, and other components necessary for the cultivation of various cell types in vitro.

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Normal human AB serum IMDM + 10% AB human serum AIM-V + 10% human AB serum Heat-inactivated human AB serum Normal human serum type AB AB Serum Human serum

119 protocols using human ab serum

Expansion of Antigen-Specific CAR-T Cells

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CAR-T cells were labeled with FITC-conjugated anti-human NGFR mAb (clone ME20.4) and isolated using anti-FITC microbeads (Miltenyi Biotec). Isolated CAR-library T cells were stimulated with the K562-based APCs established as above at an E/T ratio of 20:1^{35 (link)–37 (link)}. To expand the A2/NY-ESO-1₁₅₇-specific CAR-library T cells, A2-APCs were pulsed with 10 μg/mL NY-ESO-1₁₅₇ peptide, irradiated, and the floating peptide was removed to stimulate T cells. To expand the CD19-specific CAR-library T cells, CD19-APCs were utilized without loading any peptides. These CAR-library T cells were cultured in RPMI1640 supplemented with 50 μg/mL gentamicin and 10% human AB serum in the presence of 10 IU/mL human IL-2 and 10 ng/mL human IL-15 (PeproTech). Following three stimulations, antigen-specific CAR-library T cells were used for analyses. In some experiments, newly identified CD19 CAR-T cells as well as the original CD19 CAR-T cells were stimulated with Raji cells (E/T 1:1) or CD19-APCs (E/T 10:1). After stimulation, the proliferation activity of the CD19 CAR-T cells was assessed by calculation of the fold increase.

Ochi T., Maruta M., Tanimoto K., Kondo F., Yamamoto T., Kurata M., Fujiwara H., Masumoto J., Takenaka K, & Yasukawa M. (2021). A single-chain antibody generation system yielding CAR-T cells with superior antitumor function. Communications Biology, 4, 273.

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Expansion and Differentiation of CD34+ Cells

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CD34⁺ cells were obtained from magnetically sorted mononuclear samples of G-CSF–mobilized peripheral blood from donors and were frozen after isolation. Cells were obtained from the Fred Hutchinson Cancer Research Center, Seattle, USA or the Department of Hematology/Oncology Flow Cytometry Research Facility at Boston Children’s Hospital. Cells were thawed and washed into PBS with 1% human AB serum (Atlanta Biologicals), pelleted and then seeded in differentiation medium containing IMDM with 2% human AB plasma, 3% human AB serum, 1% P/S, 200 μg/mL holo-transferrin, 10 ng/mL SCF (PeproTech, Inc.), 1 ng/mL IL-3 (PeproTech, Inc.) and 3 U/mL erythropoietin (EPO) (Amgen). Where an expansion phase is indicated, CD34⁺ cells were cultured in StemSpan SFEM II medium (STEMCELL Technologies) supplemented by 1X CC100 (containing FLT3 ligand, stem cell factor (SCF), IL-3, and IL-6, STEMCELL Technologies) for 5 days prior to differentiation. Cells were maintained at a density between 0.1 × 10⁶ and 0.5 × 10⁶ cells per milliliter, with medium changes every other day as necessary. Cells were incubated at 37 °C with 5% CO₂.

Khajuria R.K., Munschauer M., Ulirsch J.C., Fiorini C., Ludwig L.S., McFarland S.K., Abdulhay N.J., Specht H., Keshishian H., Mani D.R., Jovanovic M., Ellis S.R., Fulco C.P., Engreitz J.M., Schütz S., Lian J., Gripp K.W., Weinberg O.K., Pinkus G.S., Gehrke L., Regev A., Lander E.S., Gazda H.T., Lee W.Y., Panse V.G., Carr S.A, & Sankaran V.G. (2018). Ribosome Levels Selectively Regulate Translation and Lineage Commitment in Human Hematopoiesis. Cell, 173(1), 90-103.e19.

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Purification and Phenotypic Analysis of AML Cells

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The leukemic samples were thawed and CD34⁺ leukemic cells were purified using a Lineage Cell Depletion kit (Miltenyi Biotec; Lund, Sweden) and CD14⁺ monocytic AML cells were isolated using BD IMag CD14 Magnetic Particles (BD Biosciences) according to the manufacturer’s instructions. The purity of the CD34⁺ and CD14⁺ populations was consistently >96 and >98%, respectively, as judged by flow cytometry. The purified cells were cultured in IMDM supplemented with 10% human AB serum and IL-4 (600 U/ml) and GM-CSF (500 U/ml) (both from Peprotech; Stockholm, Sweden) in the presence or absence of HDC at a concentration (100 µM) that optimally saturates histamine H₂ receptors (H₂R) (25 (link)). After 5 days in culture, cells were analyzed by flow cytometry for expression of maturation markers. Purified CD14⁺ monocytes or CD3⁺ T cells (negative control) from three AML patients with FAB-M4 AML were analyzed for malignant markers using fluorescent in situ hybridization or PCR as described in Ref. (11 (link)). These analyses verified that the monocytes belonged to the malignant clone. A portion of each sample was refrozen and analyzed at a later time point for H₂R, NOX2, FPR1, and FPR2 expression by flow cytometry. One patient sample, containing less than 1% viable leukemic cells after the process of refreezing, was excluded from this analysis.

Kiffin R., Grauers Wiktorin H., Nilsson M.S., Aurelius J., Aydin E., Lenox B., Nilsson J.A., Ståhlberg A., Thorén F.B., Hellstrand K, & Martner A. (2018). Anti-Leukemic Properties of Histamine in Monocytic Leukemia: The Role of NOX2. Frontiers in Oncology, 8, 218.

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Human CD8+ T Cell Suppression Protocol

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This study was approved by the Ethics Committee of Shanghai Blood Center (Permit Number: SBC-IRB-2013-07). All human donors were enrolled in the study after providing written informed consent. Human CD8⁺T cells were derived from peripheral blood mononuclear cells (PBMCs) collected from healthy blood donors (Shanghai Blood Center, Shanghai, China) and sorted using a human CD8⁺T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, 1 × 10⁵/mL hCD8⁺T cells were induced with TGF-β1 (50 ng/mL, PeproTech) and RAPA (100 nM, LC Laboratories) plus anti-CD3/CD28-coated expansion beads (cells : beads = 1 : 4, Invitrogen) and IL-2 (1600 IU/mL, PeproTech) in 1% human AB serum (from healthy blood donors, Shanghai Blood Center, China) in RPMI 1640. Cells were cultured in 96-well round plates and harvested after 9 days.

Sun J., Yang Y., Huo X., Zhu B., Li Z., Jiang X., Xie R., Gao L., Sun Y., Fan H., Zhu Y, & Yang J. (2019). Efficient Therapeutic Function and Mechanisms of Human Polyclonal CD8+CD103+Foxp3+ Regulatory T Cells on Collagen-Induced Arthritis in Mice. Journal of Immunology Research, 2019, 8575407.

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Monocyte-Derived Macrophage Differentiation

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LRS chambers were obtained from healthy anonymous blood bank donors. PBMCs were isolated using Ficoll-Paque (GE Healthcare Life Sciences) density gradient separation. Monocytes were isolated by plating ~1 × 10⁸ PBMCs in T-25 flasks with serum-free RPMI and incubating for 1 h, followed by 3x rigorous washes with DPBS (PBS +Ca +Mg) to remove non-adherent cells and ensuring that the final wash results in clear supernatant. The media was replaced with IMDM supplemented with 10% Human AB Serum (Gemini), and the cells were allowed to differentiate into macrophages over 7–9 days. To generate M2 macrophages, the media was replaced on day 4 with IMDM supplemented with 10% Human AB Serum, 20 ng/mL IL-4 (Peprotech), 50 ng/mL IL-10, and 50 ng/mL TGF-β. No media changes were conducted for M0 macrophages throughout the differentiation period.

Morimoto M., Till N.A, & Bertozzi C.R. (2023). Tumor Immune Cell Targeting Chimeras (TICTACs) For Targeted Depletion of Macrophage-Associated Checkpoint Receptors. bioRxiv.

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HCMV Infection and Cell-Mediated Cytotoxicity

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HFFs were seeded in 96-wells plates and infected with GFP-HCMV (AD169) at a MOI of 0.4 for 2 h at 37°C. Cells were washed twice with serum-free DMEM and incubated for 24 h in supplemented DMEM. HeLa cells were transfected with pcDNA3.1-HA-IE1/2, pcDNA3.1-HA-IE1-FLAG-HA, pcDNA3.1-IE1-FLAG-HA, or empty vector combined with a GFP plasmid using PEI for 20 h. LAK cells were obtained by culturing PBMCs for 4 days in RPMI 1640 medium (Gibco) supplemented with 5% human AB serum, 2.5% sodium bicarbonate (Gibco) and 1000 units/ml of recombinant interleukin-2 (WOKA, Japan). Non-autologous LAK or NK cells (YT-Indy) were added at indicated E:T ratios for 6 h co-culture at 37°C. ZVAD-fmk was added prior to addition of effector cells. Cells were washed twice with PBS and directly lysed in reducing sample buffer for immunoblotting analysis.

Shan L., Li S., Meeldijk J., Blijenberg B., Hendriks A., van Boxtel K.J., van den Berg S.P., Groves I.J., Potts M., Svrlanska A., Stamminger T., Wills M.R, & Bovenschen N. (2020). Killer cell proteases can target viral immediate-early proteins to control human cytomegalovirus infection in a noncytotoxic manner. PLoS Pathogens, 16(4), e1008426.

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Mosquito Infection with Arboviruses

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Three- to five-day old female mosquitoes were starved for 24 h and fed on an infectious blood meal containing 40% volume of washed erythrocytes from specific pathogen free (SPF) pig’s blood (Prestige BioResearch, Singapore), 5% 10 mM ATP (Sigma-Aldrich, USA), 5% human serum (Corning human AB serum, USA) and 50% virus solution in RPMI media (Gibco, USA), using Hemotek membrane feeder system (Discovery Workshops, UK). The virus titers in blood meals were 2 × 10⁷ pfu/ml for DENV2, 6 × 10⁶ pfu/ml for ZIKV, and 1.5 × 10⁸ pfu/ml for CHIKV, which all resulted in 100% SG infection^{15 (link)}. Bloodmeal titers were validated by plaque assay using BHK-21 cells. Control mosquitoes were fed with the same blood meal composition except for the virus solution, which was replaced by RPMI media. Following oral feeding, fully engorged females were selected and kept in a cage with ad libitum access to a 10% sucrose solution in an incubation chamber with conditions similar to insect rearing.
For inoculation, mosquitoes were cold-anesthetized and intrathoracically injected with 0.5 pfu of either DENV2, ZIKV or CHIKV using a Nanoject-II (Drummond scientific company, USA). The same volume of RPMI media was injected as control. Virus inoculation was conducted four days post dsRNA injection.

Chowdhury A., Modahl C.M., Missé D., Kini R.M, & Pompon J. (2021). High resolution proteomics of Aedes aegypti salivary glands infected with either dengue, Zika or chikungunya viruses identify new virus specific and broad antiviral factors. Scientific Reports, 11, 23696.

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Peripheral Blood Mononuclear Cell Stimulation

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Heparinized peripheral blood was layered over an equal volume of Histopaque 1077 (Sigma-Aldrich; St. Louis, MO) and centrifuged per the manufacturer’s instructions. Mononuclear cells were collected from the interface between Histopaque and plasma. PBMCs were washed and resuspended in RPMI 1640 media (Gibco/Life Technologies; Grand Island, NY) containing 10% heat-inactivated human AB serum, penicillin (100 U/ml), and streptomycin (100 μg/ml) (Gibco/Life Technologies). Cells were cultured in 96-well round-bottom plates (1.3 to 3.3×10⁶ cells/ml) at 37°C with 5% CO₂ for 7 days with IL-33 or IL-25 at the indicated concentrations in the presence or absence of IL-2 (20 U/ml), IL-7 (10 ng/ml), or TSLP (10 ng/ml). IL-5, IL-13, and IFNγ concentrations in the cell-free supernatants were determined using ELISA kits as recommended by the manufacturer (Thermo Fisher Scientific; Rockford, IL). (See additional Methods description in the Online Repository)

Bartemes K., Kephart G., Fox S.J, & Kita H. (2014). Enhanced Innate Type 2 Immune Response in Peripheral Blood from Patients with Asthma. The Journal of allergy and clinical immunology, 134(3), 671-678.e4.

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Modulation of T Cell Cytokines by CTLA4-Ig and ST6

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Human PBMCs were seeded in triplicate in 6-well plates at 1×10⁵ cells per well in 2 mL of medium. The cells were cultured in RPMI 1640 medium containing 10% human AB serum (Gibco) supplemented with 1% penicillin/streptomycin at 37°C in a 5% CO₂ atmosphere for 24 h. Then, T cells and B cells were separated using a magnetic cell separation column. The B cells were inactivated using 10-Gy γ-ray irradiation at the Korea Institute of Radiological & Medical Sciences (KIRMS, Seoul, Korea) (Walcher et al., 2021 (link)). Inactivated B cells and isolated T cells were co-cultured for 24 h and then incubated with CTLA4-Ig (5 μg/mL) or ST6 (5, 10, 20 μg/mL) for 24 h. Then, the cells were centrifuged at 500×g at 4°C for 5 min. IL-2, TNF-α, and INF-γ cytokine levels were determined using the Quantikine^® ELISA kit (R&D Systems). The absorbance at 450 nm was measured using a FlexStation^® 3 Multi-Mode Microplate Reader (Molecular Devices).

Piao Y., Yun S.Y., Kim H.S., Park B.K., Ha H.C., Fu Z., Jang J.M., Back M.J., Shin I.C., Won J.H, & Kim D.K. (2022). A Novel Therapeutic Effect of a New Variant of CTLA4-Ig with Four Antennas That Are Terminally Capped with Sialic Acid in the CTLA4 Region. Biomolecules & Therapeutics, 30(6), 529-539.

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Cryopreserved PBMC Analysis in T1D

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Cryopreserved peripheral blood mononuclear cells (PBMC) were provided by Type 1 Diabetes TrialNet from the clinical trial NCT00505375 (7 (link)). Samples from 40 abatacept- and 19 placebo-treated individuals were analysed at baseline, year 1 and year 2. Samples were provided blinded to treatment and time of sampling; each subject’s samples from the three collection times were stained and analysed on the same day. TrialNet Coordinating Centre provided clinical and demographic metadata following submission of raw data files. PBMCs were thawed at 37°C, washed in pre-warmed basal media (RPMI1640 Glutamax with HEPES, containing 100U/mL penicillin and 100μg/mL streptomycin (all Gibco)) containing 5% human heat-inactivated AB serum (Gibco), 20mM tumour necrosis factor (TNF)-α Protease Inhibitor-2 (TAPI-2) (Calbiochem) and 50U/mL Benzonase (Millipore). Cells were rested for 1 hour at 37°C/5% CO₂ in basal media containing 10% human AB serum and 20mM TAPI-2 before washing in 1X basal media and 1X DPBS (Gibco) and staining of 2×10⁶ PBMC each for flow and mass cytometry.

Eichmann M., Baptista R., Ellis R.J., Heck S., Peakman M, & Beam C.A. (2020). CO-STIMULATION BLOCKADE DISRUPTS CD4+ T-CELL MEMORY PATHWAYS AND UNCOUPLES THEIR LINK TO DECLINE IN β-CELL FUNCTION IN TYPE 1 DIABETES. Journal of immunology (Baltimore, Md. : 1950), 204(12), 3129-3138.

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