Every 3 to 4 days, supernatants were collected from the cocultures, centrifuged at 1800× g for 10 min and 4 °C, decanted to remove cell pellets, and stored at −80 °C. A total of six collections were performed, reaching 23 days of coculture. Once all partial samples were obtained, they were thawed at room temperature, mixed, and sterilized by filtration with 0.22 μm filters (Merck KGaA, Darmstadt, Germany). The final product was packaged in differentiated containers with the doses foreseen for its use. Part of the prepared doses was subjected to concentration, using Vivaspin tubes (Sartorius, Göttingen, Germany), capable of concentrating liquids. Centrifugation was carried out at 4 °C to maintain the properties of the biomolecules present for as long as necessary to achieve the desired concentration. In this way, the concentrated supernatant was adjusted to a 2.5× nd 5× concentration of the original product by diluting with the culture medium used (CTS-AIM-V™, Gibco-BRL, Waltham, MA, USA), which is the one used for PRS CK STORM.
Cts aim 5
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CTS AIM-V is a laboratory instrument designed for automated immunoaffinity sample preparation. It performs key steps in the sample preparation process, including extraction, purification, and elution, to streamline workflows and improve reproducibility. The CTS AIM-V can handle a variety of sample types and target analytes, making it a versatile tool for researchers and scientists.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Vivaspin tube, by Sartorius (1 mentions)
0.22 μm filter, by Merck Group (1 mentions)
M csf, by R&D Systems (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Aim 5, by Thermo Fisher Scientific (1 mentions)
Collagenase, by Fujifilm (1 mentions)
Lympholyte m, by Cedarlane (1 mentions)
Cts optmizer, by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Thermo Fisher Scientific (1 mentions)
Gibco macrophage sfm, by Thermo Fisher Scientific (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Penicillin streptomycin glutamine solution, by Thermo Fisher Scientific (1 mentions)
Gentamycin, by Quality Biological (1 mentions)
7 protocols using cts aim 5
1
Concentration of Supernatant from Cell Cocultures
2
Monocyte Seeding and Culture
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Monocyte seedings were changed to monocyte culture medium (CTS-AIM-V™, Gibco-BRL, Waltham, MA, USA) with 2 mL per well, and empty inserts were placed in the wells, with a small amount of medium to adjust their porous membrane properties, and incubated again for 15 min. Finally, monocytes were seeded in a final volume of 1.5 mL and supplemented with M-CSF (R&D Systems, Minneapolis, MN, USA) at a final concentration of 10 ng/mL.
3
PBMNC and Fibroblast Mixed Sheets for Wound Healing
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PBMNCs were isolated from mouse peripheral blood using Lympholyte-M (Cedarlane Laboratories Ltd., Hornsby, Ontario, Canada) and cultured in CTS AIM-V (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (Thermo Fisher Scientific). Fibroblasts were isolated from the tails of mice using collagenase (Wako, Osaka, Japan) and cultured in AIM-V (Thermo Fisher Scientific) containing 10% fetal bovine serum. For mixed cell sheets, 1 ml PBMNCs (2 × 106 cell/ml) and 1 ml fibroblasts (1.25 × 105 cell/ml) were added to the same well in UpCell 24 multiwell plates (CellSeed Inc., Tokyo, Japan). For hypoxic preconditioning, cells were incubated for 2 days under normoxic conditions (37 °C in 20% O2 and 5% CO2) and then for 1 day under hypoxic conditions (33 °C in 2% O2 and 5% CO2). Mixed cell sheets were aspirated using a 1000 µl pipet and transplanted onto magnet-implanted ulcers. The ulcers were covered with UrgoTul (Laboratories Surgo, France) and Derma Aid (ALCARE, Japan) and then secured with an elastic adhesive bandage (Nichiban, Japan).
4
Optimal Culture Conditions for Ag-Specific T-cells
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Many culture media were comparatively screened for their capacity to support propagation of natural Ag-specific T-cells from human PBMC, including RPMI 1640 (Thermo-Fisher, Waltham MA, #11875119), CTS AIM-V (Thermo-Fisher #0870112DK), CTS OpTmizer (Thermo-Fisher #A1048501), Ex Vivo10 (Lonza, Walkersville MD, #04-380Q), and Gibco macrophage SFM (Thermo-Fisher #12065074), variously supplemented with human AB serum (HuAB) already heat-deactivated at purchase (Fisher-Scientific/Gemini #100-512, Lot#H15M03A). Optimal culture performance was attained employing AIM-V already formulated to contain glutamine, penicillin and streptomycin, supplemented only with Gibco Amphotericin B at an optimized final concentration of 0.125 μg/ml (Thermo-Fisher #15290018). Serum-free T-cell propagation was not sustainable, but supplementation of AIM-V with 0.5% HuAB during Step 1 (d0-2 of culture) and 2.0% HuAB during Step 2 (d2 and thereafter) proved optimal for robust expansion of both CD4+ and CD8+, natural Ag-specific T-cells. Culture incubators were maintained at a CO2 tension of 5%.
5
Fibroblast Cell Sheet Cultivation
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The cell sheets were prepared using the previously reported method, 15, 16 5.0 £ 10 5 fibroblasts were cultured in a 24-well culture dish with 2 mL of DMEM supplemented with 10% FBS. After incubation for 24 hours, the medium was replaced with 2 mL of CTS AIM V (Thermo Fisher Scientific) and HFDM-1 (+) (Cell Science & Technology Institute, Sendai, Japan) supplemented with 5% FBS. After additional incubation for 48 hours, the cell sheets were detached using dispase.
6
Detailed PBMC Immunophenotyping Workflow
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PBMC immunophenotyping was designed, performed, and analyzed by Immune Monitoring and Genomics Facility, UNC Lineberger Comprehensive Cancer Center. Cryopreserved PBMCs were thawed in Dextran-Albumin (CSL Behring #44206-251-10) solution and washed with AIM-V CTS (Gibco #0879122DK)/5% of Human AB Serum (Gemini#100-512), following cell resuspension in 1X HBSS (Gibco #14175-095). Viable PBMC numbers were determined and distributed at 2 million cells per patient per timepoint per assay tube. Flow cytometry panels were used to analyze T cell (Supplementary Table 1 ), B cell (Supplementary Table 2 ), and myeloid (Supplementary Table 3 ) subsets. Samples were aliquoted and analyses performed separately for T, B, and myeloid cells. Intracellular staining for Foxp3 in T cell panel and Ki67 in B cell panel were performed by utilizing eBioscienceFoxp3/TF staining kit (ThermoFisher #00-5523-00). All stained cells were acquired on BD LSRFortessa (Serial# H64717700116). Gating strategy was based on fluorochrome minus one (FMO) control staining to distinguish positive and negative populations in each subset. All gating and subset analyses were done by FlowJo v8 (Supplementary Fig. 3 ). Flow cytometry values were standardized between aliquots by assessing parent population percentage. Percentages of parental populations were analyzed by paired Wilcoxon tests.
7
Primary Leukocyte Cell Culture
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Primary leukocytes were cultured in cell growth medium in the presence of indicated cytokines. Cell growth medium contains 45% RPMI 1640, 50% AIM V CTS (Gibco by Life Technologies), and 5% non-AB pooled human serum (Gemini Bio-Products) by volume and was supplemented with 65 ug/ml gentamycin (Quality Biological), and 1X penicillin-streptomycin-glutamine solution (Gibco by Life Technologies).
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