Aim 5

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, Japan, China, Sweden, United Kingdom

The AIM-V is a laboratory equipment designed for cell culture and protein production applications. It functions as a serum-free medium for the in vitro cultivation of a variety of cell types.

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Therapeutic AIM-V medium Aim V serum AIM V-AlbuMAX(BSA) AIM V-AlbuMAX medium AIM-V medium CTS™ AIM-V culture medium FBS-AIM-V medium AIM-V+AlbuMAX GIBCO Aim V™ CTS AIM-V

174 protocols using aim 5

Expansion of Engineered T Cells

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H1-CAR ILC2s were isolated from week 5–9 ATOs as described above. For proliferation assays, up to 1×10⁵ cells were plated in 200 μl AIM V (ThermoFisher Scientific, Cat. 12055091), 5% human AB serum (Gemini Bio, Cat. 100–512) with 20 ng/mL rhIL-2 (Peprotech) and 20 ng/mL rhIL-7 (Peprotech) plus indicated cytokines at 20 ng/mL (Peprotech) in the absence or presence of irradiated NALM6 cells at a 3:1 effector to target (E:T) ratio. Fresh cytokines were replenished at day 3 via half-media change, and cells were replated into larger wells when confluent, approximately every 2–3 days. Irradiated NALM6 cells were added again on day 7 of expansion. Cells were counted twice a week on a hemocytometer.
H1-CAR T cells were expanded at 5×10⁵ cells/mL in AIM V (ThermoFisher Scientific, Cat. 12055091) supplemented with 5% human AB serum (Gemini Bio, Cat. 100–512), 5 ng/ml rhIL-7 (R&D), and 100 IU/ml rhIL-2 (Miltenyi Biotec, Cat. 130–097-748) with irradiated NALM6 cells added at a 3:1 E:T ratio 5 days prior to functional assays.

Weinreich M., Liang C, & Stillman B. (1999). The Cdc6p nucleotide-binding motif is required for loading Mcm proteins onto chromatin. Proceedings of the National Academy of Sciences of the United States of America, 96(2), 441-446.

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Cell Culture Protocol for Diverse Cell Lines

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The culture media, minimum essential medium (MEM), DMEM, and RPMI (Thermo Fisher Scientific, Waltham, MA), were supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific), 2 mM glutamine (Thermo Fisher Scientific), 10 U/mL penicillin, and 10 μg/mL streptomycin (Thermo Fisher Scientific) and named shortly M10, D10, and R10, respectively. CHO cells and their transfectants were maintained in M10 supplemented with non-essential amino acid (M10-NEAA). 293FT cells were cultured in D10, and Jurkat E6.1 cells, obtained from European Collection of Authenticated Cell Cultures through DS Pharma, were maintained in R10. Human peripheral blood mononuclear cells (PBMCs) from healthy donors were prepared by Ficoll-Paque (GE Healthcare Life Sciences, Pittsburgh, PA) density gradient and cultured in AIM-V (Thermo Fisher Scientific) supplemented with 5% FBS and 10 mM HEPES (shortly, complete AIM-V) overnight to remove plastic-adherent monocytes. Monocyte-depleted PBMCs were used for transduction experiments. All cells were grown at 37°C with 5% CO₂.

Nunoya J.I., Masuda M., Ye C, & Su L. (2019). Chimeric Antigen Receptor T Cell Bearing Herpes Virus Entry Mediator Co-stimulatory Signal Domain Exhibits High Functional Potency. Molecular Therapy Oncolytics, 14, 27-37.

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Expansion and Cryopreservation of T Cells

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PBMCs were isolated from leukapheresis units using Ficoll density gradient separation (GE Healthcare, Chicago, IL, USA) and cryopreserved. PBMCs were thawed and cultured in AIM-V (Thermo Fisher Scientific, Waltham, MA) supplemented with 5% CTS^TM Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific, Waltham, MA) and human IL-2 (300 IU/ml, Quangang, Shandong, China). PBMCs were activated and expanded with CTS^TM Dynabeads^TM CD3/CD28 (Thermo Fisher Scientific, Waltham, MA) for 24 h. T cells were transduced with LVs at multiplicity of infection (MOI) of 1. Three days after transduction, the cells were extensively washed to remove the LV particles. T cells were further expanded in AIM-V medium supplemented with 5% CTS^TM Immune Cell SR (Thermo Fisher Scientific, Waltham, MA) for three to ten days in the presence of IL-2 (300 IU/ml, Quangang). The cell product was washed twice with normal saline (Qidu pharmaceuticals, Shandong, China) containing 2.5 % human serum albumin (CSL Behring, King of Prussia, PA, USA). Cells were resuspended in Cryostor^® CS10 Cell Freezing Medium (STEMCELL Technologies, Vancouver, Canada) for cryopreservation and stored in liquid nitrogen.

Zhang X., Wang T., Zhu X., Lu Y., Li M., Huang Z., Han D., Zhang L., Wu Y., Li L., Klawonn F, & Stripecke R. (2023). GMP development and preclinical validation of CAR-T cells targeting a lytic EBV antigen for therapy of EBV-associated malignancies. Frontiers in Immunology, 14, 1103695.

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Real-Time Cytotoxicity Monitoring

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Cytotoxicity was measured in a real-time cell analyzer xCELLigence (Roche). Adherent tumor cell lines were incubated for 24 hours in the xCELLigence 96-well plates in the respective tumor cell media. Cell culture medium was removed, cells were washed with AIM-V (Thermo Fisher Scientific), and test molecules and effector cells were added in the respective concentrations in AIM-V medium. Readout was performed over 7 to 48 hours in triplicates. For measurement of lactate dehydrogenase (LDH) release, supernatants of cytotoxicity assays were analyzed with the Cytotoxicity Detection Kit (Roche) according to the manufacturer's protocol.
Absorption was detected at 490 nm using Tecan Sunrise Reader (Tecan). Spontaneous release is defined as cell index from target cells and effector cells only, specific release from cell index of each specimen. Specific lysis in percent (%) is calculated as [(cell index spontaneous release À cell index specimen)/(cell index spontaneous release)] Â 100. Maximum release was determined through addition of 1% Triton X-100 (Sigma-Aldrich) to target cells.

Schmittnaegel M., Hoffmann E., Imhof-Jung S., Fischer C., Drabner G., Georges G., Klein C, & Knoetgen H. (2016). A New Class of Bifunctional Major Histocompatibility Class I Antibody Fusion Molecules to Redirect CD8 T Cells. Molecular cancer therapeutics, 15(9).

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Cryopreservation and Thawing of PBMCs

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PBMC were isolated by Ficoll-Hypaque density gradient method, cells were washed and suspended in 20% fetal calf serum (FCS) in AIM-V (Invitrogen, Carlsbad, CA) and kept cool on ice, counted and frozen using a cold freshly prepared freezing medium composed of 20% FCS, 20% dimethyl sulphoxide (DMSO) in AIM-V. Cells were kept at −80°C for 2–3 days and transferred to liquid nitrogen until use. During thawing, cells were transported in liquid nitrogen to a water bath (37°C) for 30 to 40 seconds until thawed half way and resuspended in 10% FCS in AIM-V (37°C) containing 1/10,000 benzonase until completely thawed, washed 2 times (5–7 minutes each) and counted. The percentage viability obtained was >75% and cells were incubated with anti-CD25 magnetic beads or used for FACS analysis.

Bobosha K., Wilson L., van Meijgaarden K.E., Bekele Y., Zewdie M., van der Ploeg- van Schip J.J., Abebe M., Hussein J., Khadge S., Neupane K.D., Hagge D.A., Jordanova E.S., Aseffa A., Ottenhoff T.H, & Geluk A. (2014). T-Cell Regulation in Lepromatous Leprosy. PLoS Neglected Tropical Diseases, 8(4), e2773.

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NK Cell Degranulation Assay

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NK cells were first washed with 1x PBS (Hyclone) once and re-suspended into 1 mL AIM-V (Invitrogen) supplemented with 5% AB serum (Valley Biomedical). APC-conjugated CD107a antibody (BD Biosciences) and Golgi stop containing monensin (BD Biosciences) were added in and mixed up well with the cells. Target cells were washed and re-suspended into AIM-V (Invitrogen) supplemented with 5% AB serum (Valley Biomedical), and subsequently seeded into a U-bottom 96-well plate (Thermo Fisher Scientific) at the concentration of 2 × 10⁵ cells/100 μL/well. NK cells were then co-cultured at a 1:1 E: T ratio with target cells in a 37°C, 5% CO₂ incubator for 5 h. After incubation, the cells were used for anti-CD56 PE antibody staining (Miltenyi). The cells were then suspended in 150 μL autoMACS Running Buffer (Miltenyi) for the detection of the CD107a surface expression on NK cells by BD Accuri C6 Flow Cytometer (BD Biosciences).

Du Z., Ng Y.Y., Zha S, & Wang S. (2021). piggyBac system to co-express NKG2D CAR and IL-15 to augment the in vivo persistence and anti-AML activity of human peripheral blood NK cells. Molecular Therapy. Methods & Clinical Development, 23, 582-596.

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Expansion and Rapid Expansion of Tumor-Infiltrating Lymphocytes

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The tumor samples were cut into 1-3 mm² fragments and placed in TIL culture media [TIL-CM: RPMI-1640 with GlutaMax (Gibco/Invitrogen), 1× Pen–Strep (Gibco/Invitrogen), 50 μmol/L 2-mercaptoethanol (Gibco/Invitrogen), 20 μg/mL Gentamicin (Gibco/Invitrogen), and 1 mmol/L pyruvate (Gibco/Invitrogen] with 6000 IU/ml IL-2 in 24-well plates for a period of 4 weeks, as previously described (17 (link),29 (link)). The same method was applied for the metastatic melanoma samples. For the 4-1BB condition, both 6000 IU/ml IL-2 and 10 ug/ml 4-1BB mAb were added in the culture plates on day 0 and day 4 or 5. TIL were expanded for up to 35 days prior to performing the described assays or the rapid expansion protocol (REP). The REP was performed in the G-Rex 10 device (Wilson Wolf Manufacturing) following a scaled-down version of the previously described protocol (29 (link)). Briefly, TIL were put in culture with pooled allogeneic irradiated PBMC feeder cells at a ratio of 1 TIL to 200 feeders in combination with 6000U/ml IL-2 and 30ng/mL of anti-CD3 (OKT3 clone) on day 0 of the REP. The REP process lasted for 14 days, with REP-CM (half TIL-CM and half AIM-V (Invitrogen)) used for the first 7 days and only AIM-V for the last 7 days of expansion.

Sakellariou-Thompson D., Forget M.A., Creasy C., Bernard V., Zhao L., Kim Y.U., Hurd M.W., Uraoka N., Parra E.R., Kang Y., Bristow C.A., Rodriguez-Canales J., Fleming J.B., Varadhachary G., Javle M., Overman M.J., Alvarez H.A., Heffernan T.P., Zhang J., Hwu P., Maitra A., Haymaker C, & Bernatchez C. (2017). 4-1BB agonist focuses CD8+ tumor-infiltrating T-cell growth into a distinct repertoire capable of tumor recognition in pancreatic cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(23), 7263-7275.

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T Cell Subset Analysis and Treg Suppression Assay

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T helper cell subsets were assessed by intracellular flow cytometry on PBMCs stimulated in 96-well round bottom plates with PMA and ionomycin for 4 h. T regulatory cell induction was done by culturing CD4⁺CD25⁻ isolated T cells in serum-free medium (Aim V, Invitrogen) in 96-well round bottomed cell culture plates with plate bound anti-CD3ε (UCHT) with soluble anti-CD28 for 120 h with IL-2 (100 IU/ml) and TGF-β1 (10 ng/ml) (antibodies and cytokines used are from R&D Systems, Abingdon, UK) for 5 days and then harvested to measure their maturation as induced Tregs (iTregs) by FACS. Their suppressive capacity was assessed as previously described (30 ) after harvest by co-culturing Tregs with CFSE labeled allogeneic PBMC’s and super antigen-pulsated Epstein–Barr transformed B cells (EBV-B cells) in Aim V serum-free medium (Invitrogen). Staphylococcal enterotoxins (SEA, SEB, and SEE, Toxin Technologies, 1 µg/ml of each) were used for 2 h pulses before washing three times with PBS. The PBMCs: EBV-B cells and iTregs: EBV-B cells ratio was constant at 10:1 while the iTregs: PBMCs ratio varied from 1:1 to 1:32. The cells were harvested after 3 days and the suppressive capacities of iTregs assessed by flow cytometric analysis by proliferation calculations.

Lemarquis A.L., Einarsdottir H.K., Kristjansdottir R.N., Jonsdottir I, & Ludviksson B.R. (2018). Transitional B Cells and TLR9 Responses Are Defective in Selective IgA Deficiency. Frontiers in Immunology, 9, 909.

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Purification and Characterization of Mature CD8+ T Cells

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Mature CD8SP cells from PSC-ATOs were isolated by magnetic negative selection using the CD8+ T Cell Isolation Kit
(Miltenyi, Cat. 130–096-495) and sorted by FACS to further deplete CD45RO+ cells (containing immature CD8SP T cells and
DN/4ISP T cell precursors). Purified T cell populations were plated in 96-well U-bottom plates in 200 µl AIM V
(ThermoFisher Scientific, Grand Island, NY) with 5% human AB serum (Gemini Bio-Products, West Sacramento, CA).
PMA/ionomycin/protein transport inhibitor cocktail or control protein transport inhibitor cocktail (eBioscience, San Diego,
CA) were added to each well and incubated for 6h. Cells were washed and stained for CD3, CD4, and CD8 (Biolegend, San Diego,
CA) and Zombie UV™ Fixable Viability dye (Biolegend, San Diego, CA) prior to fixation and permeabilization with an
intracellular staining buffer kit (eBioscience, San Diego, CA) and intracellular staining with antibodies against IFNγ,
TNFα, and IL-2 (Biolegend, San Diego, CA).

Montel-Hagen A., Seet C.S., Li S., Chick B., Zhu Y., Chang P., Tsai S., Sun V., Lopez S., Chen H.C., He C., Chin C.J., Casero D, & Crooks G.M. (2019). Organoid-induced differentiation of conventional T cells from human pluripotent stem cells. Cell stem cell, 24(3), 376-389.e8.

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Intracellular Cytokine Profiling of Mature T Cells

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Mature CD8SP or CD4SP cells from ATOs were isolated by magnetic negative selection using the CD8+ or CD4+ Isolation Kits (Miltenyi) and sorted by FACS to further deplete CD45RO+ cells (containing immature naïve T cells and CD4ISP precursors). Purified T cell populations were plated in 96-well U-bottom plates in 200 μl AIM V (ThermoFisher Scientific, Grand Island, NY) with 5% human AB serum (Gemini Bio-Products, West Sacramento, CA). PMA/ionomycin/protein transport inhibitor cocktail or control protein transport inhibitor cocktail (eBioscience, San Diego, CA) were added to each well and incubated for 6h. Cells were stained for CD3, CD4, and CD8 (Biolegend, San Diego, CA) and UV455 fixable viability dye (eBioscience, San Diego, CA) prior to fixation and permeabilization with an intracellular staining buffer kit (eBioscience, San Diego, CA) and intracellular staining with antibodies against IFNγ, TNFα, IL-2, IL-4, or IL-17A (Biolegend, San Diego, CA).

Seet C.S., He C., Bethune M.T., Li S., Chick B., Gschweng E.H., Zhu Y., Kim K., Kohn D.B., Baltimore D., Crooks G.M, & Montel-Hagen A. (2017). Generation of mature T cells from human hematopoietic stem/progenitor cells in artificial thymic organoids. Nature methods, 14(5), 521-530.

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