Aim 5 medium

Manufactured by Thermo Fisher Scientific

Sourced in United States, Japan, United Kingdom, Germany, Sweden, Italy, Canada, France, Netherlands, Switzerland

AIM-V medium is a serum-free, animal component-free cell culture medium designed for the growth and maintenance of a variety of cell types, including lymphocytes and T cells. It is a balanced salt solution that provides the necessary nutrients, vitamins, and growth factors to support cell growth and proliferation.

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Gibco’s serum-free AIM-V medium Complete AIM-V medium AIM V-AlbuMAX medium Therapeutic AIM-V medium GIBCOTM AIM V Medium liquid FBS-AIM-V medium AIM-V cell culture medium AIM-V medium CTS™ AIM V® Serum Free Medium AIM-V culture medium

457 protocols using aim 5 medium

Electroporation of Activated PBMCs

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PBMCs were collected under informed consent from healthy donors. PBMCs were stimulated with 600 U/ml IL-2 (rIL-2; R&D Systems, Minneapolis, MN) and 50 ng/ml anti-CD3 (OKT-3; eBioscience, San Diego, CA) in AIM-V medium (Life Technologies, Carlsbad, CA) supplemented with 2% human AB serum for 7 days. The concentration of rIL-2 was increased to 1,000 IU/ml on day 8.
Electroporation were performed with the Nucleofector device II (Lonza, Cologne, Germany). 10 × 10⁶ PBMCs were activated for 8 days as described above and were thereafter re-suspended in 100 μL of Cell Line Nucleofector Solution V (Lonza, Cologne, Germany) and TCR mRNA was added at 200 μg/ml47 (link). The mixture was placed in a certified cuvette (Lonza, Cologne, Germany) and electroporated. After electroporation, cells were re-suspended in AIM-V medium (Life Technologies, Carlsbad, CA) supplemented with 2% human AB serum and 100 IU/ml rIL-2. Transfected cells were maintained in a humidified 37 °C and 5% CO₂ incubator until flow cytometry analysis and/or co-culture experiments.

Holmström F., Chen M., Balasiddaiah A., Sällberg M., Ahlén G, & Frelin L. (2016). Functional differences in hepatitis C virus nonstructural (NS) 3/4A- and 5A-specific T cell responses. Scientific Reports, 6, 24991.

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Immunogenicity Evaluation of Peptide 2c

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The immunogenicity test of 2c was conducted by evaluated the ability of 2c to induce T cell proliferation in peripheral blood mononuclear cells (PBMC) from 50 Chinese individuals (obtained from LDEBiO, Guangzhou, China), using previous described method.^{42 (link)} Briefly, PBMC were cultured in AIMV medium (Life Technologies, Carlsbad, CA) and added to the 24-well plates (2 mL) to reach a final concentration of ∼3 ×10⁶ cells per mL, and then stimulated by addition of lixisenatide, semaglutide and 2c in AIMV medium with a final concentration of 30 μg mL⁻¹ of each tested peptides. The 24-well plates were incubated in CO₂ incubator (5%) at 37 °C for 8 days. The cells in each well of the plate were transferred to 96-well plate on days 5, 6, 7 and 8. The cultures were pulsed with [³H]-Thymidine (PerkinElmer) and incubated for further 18 h and counts per minute (cpm) for each well were determined. The stimulation index (SI) was calculated by dividing the proliferative response of the test well (cpm) by the proliferative response of the medium-only treated well (cpm) for each donor, and SI greater than 2.0 was considered positive. The % donors responding was calculated by taking the number of donors that had a positive response over the entire time course (5–8 days) as a percentage of the total number of donors that were tested.

Tang C., Li Q., Deng X., Wu W., Liao L., Liang K., Huo R., Li C., Han J., Tang W, & Jiang N. (2020). Discovery of lixisenatide analogues as long-acting hypoglycemic agents using novel peptide half-life extension technology based on mycophenolic acid. RSC Advances, 10(20), 12089-12104.

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Monocyte-Derived Dendritic Cell Generation

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Monocyte-derived DCs were generated as previously described with minor modifications (9 (link)). CD14+ monocytes were re-suspended in serum-free AIM-V medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin and placed in 24-well plates (Corning, Inc., Corning, NY, USA) for incubation at 37°C in a humidified atmosphere containing 5% CO₂ for 2 h. Following complete aspiration of the supernatant, fresh AIM-V medium supplemented with GM-CSF (1,000 IU/ml) and IL-4 (500 IU/ml; both PeproTech, Inc., Rocky Hill, NJ, USA) was added to the cells. The cells were supplied every 2 days with fresh medium. On day 5, imDC were harvested and cultured in the presence of IL-1β, IL-6, TNF-α (1,000 IU/ml; PeproTech, Inc.) and PGE2 (1 µg/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for a further 24 or 48 h, respectively, to obtain mature DCs (mDC). Supernatants were collected and retained for cytokine analysis.

Wu X., Xu F., Liu J, & Wang G. (2017). Comparative study of dendritic cells matured by using IL-1β, IL-6, TNF-α and prostaglandins E2 for different time span. Experimental and Therapeutic Medicine, 14(2), 1389-1394.

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Generation of Dendritic Cells from PBMCs

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Peripheral blood was collected from healthy HLA*A0201⁺ donors, and PBMCs were separated using lymphocyte separation medium (ICN Biochemicals, Aurora, VA), according to the manufacturer’s instructions. DCs were generated using a modification of a previously described procedure [46 (link)]. PBMCs were resuspended in AIM-V medium (Invitrogen, Carlsbad, CA) and allowed to adhere in a 6-well plate for 2 h. Adherent cells were cultured for 5 days in AIM-V medium containing 100 ng/ml GM-CSF and 20 ng/ml IL-4 (PeproTech, Rocky Hill, NJ). The culture medium was replenished every 3 days.

Tsang K.Y., Fantini M., Fernando R.I., Palena C., David J.M., Hodge J.W., Gabitzsch E.S., Jones F.R, & Schlom J. (2017). Identification and characterization of enhancer agonist human cytotoxic T-cell epitopes of the human papillomavirus type 16 (HPV16) E6/E7. Vaccine, 35(19), 2605-2611.

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T-cell Proliferation Modulation by Fatty Acids

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PBMCs were labeled with Cell Proliferation Dye (CPD) eFluor450 (Thermo Fisher Scientific) and stimulated for 4 days with plate-bound anti-CD3 (clone OKT3; Thermo Fisher Scientific). In some experiments, cells were precultured in AIM-V medium (Thermo Fisher Scientific) supplemented with BSA (10% v/v; Sigma-Aldrich) for 1 day in the absence or presence of palmitic acid (PA; 300 μM; Sigma-Aldrich), and in other experiments, cells were precultured in AIM-V medium (Thermo Fisher Scientific) without BSA supplementation for 2 days in the absence or presence of rosiglitazone (40 μM; Sigma-Aldrich). Proliferation was measured using flow cytometry to quantify the dilution of CPD. Sample size calculation, based on available data for T-cell proliferation (5 (link)), suggested a size of 10 individuals per group to detect a 50% difference in CPD low (i.e. proliferating) cells between middle-aged and old individuals with a power of 80% using a one-sided significance level of 5%. To assess the effect of fatty acids on resting cells, PBMCs were cultured up to 48 hr in AIM-V medium supplemented with 10% BSA in the absence or in the presence of palmitic acid (300 or 3000 μM).

Nicoli F., Cabral-Piccin M.P., Papagno L., Gallerani E., Fusaro M., Folcher V., Dubois M., Clave E., Vallet H., Frere J.J., Gostick E., Llewellyn-Lacey S., Price D.A., Toubert A., Dupré L., Boddaert J., Caputo A., Gavioli R, & Appay V. (2022). Altered basal lipid metabolism underlies the functional impairment of naive CD8+ T cells in elderly humans. Journal of immunology (Baltimore, Md. : 1950), 208(3), 562-570.

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T-cell Proliferation Modulation by Fatty Acids

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Investigating T Cell Proliferation Modulation

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PBMCs were labeled with Cell Proliferation Dye (CPD) eFluor450 (Thermo Fisher Scientific) and stimulated for 4 days with plate-bound anti-CD3 (clone OKT3; Thermo Fisher Scientific) in the absence or presence of CTAB (1 μM; Sigma-Aldrich). In some experiments, cells were precultured in AIM-V medium (Thermo Fisher Scientific) supplemented with bovine serum albumin (BSA; 10% v/v; Sigma-Aldrich) for 1 day in the absence or presence of palmitic acid (300 μM; Sigma-Aldrich), and in other experiments, cells were precultured in AIM-V medium (Thermo Fisher Scientific) without BSA supplementation for 2 days in the absence or presence of rosiglitazone (40 μM; Sigma-Aldrich). Proliferation was measured using flow cytometry to quantify the dilution of CPD.

Nicoli F., Cabral-Piccin M.P., Papagno L., Gallerani E., Folcher V., Dubois M., Barrett A.J., Vallet H., Frere J.J., Gostick E., Llewellyn‐Lacey S., Price D.A., Toubert A., Boddaert J., Caputo A., Gavioli R., & Appay V. (2020). Altered basal lipid metabolism underlies the functional impairment of naive CD8⁺ T cells in elderly humans.

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Fin Cell Differentiation under Culture Conditions

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Fin cells from established S. cirrhifer cell line were evaluated their differentiation under various culture conditions. 4.0×10 5 cells were seeded in L-15 media containing 10% FBS in a 25 cm 2 Collagen I coated flask or Non-coated flask (Thermo Fisher Scientific). 24 h later, the cells were washed with PBS and treated with different media. For those seeded in Collagen I coated flask, AIM V Medium (Thermo Fisher Scientific) + 10% FBS, AIM V Medium (Thermo Fisher Scientific) only, L-15 + 10% heat-inactivated SeaGrow (EastCoast Biologics, North Berwick, ME), KBM Neural Stem Cell medium (Kohjin Bio Co. Ltd., Saitama, Japan) + 1X Neural Induction Supplement (Thermo Fisher Scientific). For the cells seeded in Non-coated flask, L-15 only.
Cell differentiation was photographed every 60 seconds and the time-lapse movies were created with Axio Vision ver. 4.8 software (Carl Zeiss).
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Tsuruwaka Y., & Shimada E. (2020). Differentiation potential of fish fins –Effective utilization of the fins as food wastes–.

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CD69 Downregulation Assay for Exosome Function

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CD4+ T cells were isolated from PBMC by negative selection using AutoMACS as previously described (13 (link)). The purity of CD4+ T cells was always >95% as determined by flow cytometry. The CD69 down-regulation assay was performed as described by Muller et al. (14 (link)). Briefly, CD4+ T cells were activated with anti-CD3/anti-CD28 beads (Miltenyi, San Diego, CA, USA) at the 1:2 beads to cell ratio and IL-2 (150 U/mL, Peprotech) for 2 h. Exosomes (50 µL aliquots of SEC fraction #4) were added to activated T cells and incubated for 40 h in AIMV medium (Life Technologies, Pittsburgh, PA, USA) at 37°C. The percentages of CD69⁺ live T cells or MFI for CD69 expression levels were measured by flow cytometry after staining with CD69-FITC (BD Bioscience, San Jose, CA, USA), CD4-PE (Beckman Coulter) and 7-AAD (BD Bioscience). As controls, matching isotype Abs, non-activated T cells+PBS and activated T cells+normal control (NC) exosomes were tested in parallel.

Hong C.S., Funk S., Muller L., Boyiadzis M, & Whiteside T.L. (2016). Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer. Journal of Extracellular Vesicles, 5, 10.3402/jev.v5.29289.

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Expansion and Characterization of NLV-Specific CD8 T Cells

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Human NLV-specific CD8 T-cell lines were expanded from purified CD8 T cells of HLA-A2.1⁺ HCMV-seropositive healthy donors by stimulation with pp65 (UL83)_495-503 NLV peptide (10⁻⁶ M)-loaded and irradiated (35 Gy) autologous peripheral blood mononuclear cells (PBMC) over 2 weeks at the T cell-to-PBMC ratio of 1:1 in AIM-V medium (Life Technologies). AIM-V was supplemented with 10% human serum, recombinant human interleukin (rhIL)-2 (50 IU/mL; Proleukin, San Diego, CA, USA), rhIL-7, and rhIL-15 (each 5 ng/mL; R&D Systems) (AIM-V^cytokine). A murine NLV-specific CD8 T-cell line was generated and weekly restimulated as previously described [38 (link)]. MEF were infected with mCMV under conditions of centrifugal enhancement of infectivity [104 ]. HFF were infected with HCMV-RVKB6 and—RVKB15 for 24h at an MOI of 5. Standard 4h [⁵¹Cr]-release and 20h interferon (IFN)-γ ELISpot-assays were performed in duplicates as reported [109 (link),110 (link)]. Dose-escalating equilibrium tetramer binding data were plotted in Scatchard analysis of mean fluorescence intensity (MFI)/concentration of tetramer against MFI. The dissociation constant K_D equals -1/slope [44 (link),45 (link)].

Thomas S., Klobuch S., Podlech J., Plachter B., Hoffmann P., Renzaho A., Theobald M., Reddehase M.J., Herr W, & Lemmermann N.A. (2015). Evaluating Human T-Cell Therapy of Cytomegalovirus Organ Disease in HLA-Transgenic Mice. PLoS Pathogens, 11(7), e1005049.

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