Mo-DCs were prepared from healthy donor-derived PBMCs, as already described [23 (link)]. PBMCs were cultured with CellGro DC Medium (CellGenix GmbH, Freiburg, Germany) at 10 × 106 cells/mL for 2 h after which all cells that had not adhered to the plastic were removed from the culture. Adherent cells were cultured in CellGro DC Medium added with 1000 IU/mL of recombinant human (rh) interleukin (IL)-4 and rh-granulocyte-macrophage colony-stimulating factor (GM-CSF; CellGenix GmbH). On day 7 the culture medium was discarded and the cells were incubated in CellGro DC Medium added with IL-6 (2000 UI/mL), tumor necrosis factor-α (TNFα; 20 ng/mL), IL-1β (20 ng/mL) (all from CellGenix GmbH), and prostaglandin E2 (PGE2; 1 μg/mL) (Cayman Chemical, Ann Arbor, MI, USA). On day 9 mDCs were collected, washed, resuspended in sterile saline solution, and counted under a light microscope to assess their vitality and purity. DCs were cryopreserved in 90% autologous plasma and 10% DMSO solution until use.
Cellgro dc medium
Manufactured by CellGenix
Sourced in Germany, United States
CellGro DC medium is a serum-free and animal component-free cell culture medium designed for the differentiation and expansion of dendritic cells from monocytes.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 4 (il 4), by R&D Systems (2 mentions)
Protein low bind tubes, by Eppendorf (2 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by PAN Biotech (1 mentions)
Human serum, by Atlanta Biologicals (1 mentions)
2 3 cgamp, by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by Miltenyi Biotec (1 mentions)
Rpmi 1640, by Corning (1 mentions)
Easysep human na ve cd8 t cell isolation kit, by STEMCELL (1 mentions)
Il 1β, by CellGenix (1 mentions)
Interleukin 6 (il 6), by CellGenix (1 mentions)
Prostaglandin e2 pge2, by Cayman Chemical (1 mentions)
Clinimacs system, by Miltenyi Biotec (1 mentions)
Bactec system, by BD (1 mentions)
Anti cd28, by BD (1 mentions)
Il 1β, by R&D Systems (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Gm csf, by R&D Systems (1 mentions)
Interleukin 6 (il 6), by R&D Systems (1 mentions)
Interleukin 4 (il 4), by CellGenix (1 mentions)
17 protocols using cellgro dc medium
1
Monocyte-Derived Dendritic Cell Generation
2
Generation and Characterization of Dendritic Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dendritic cells (Ctse+/+ and Ctse−/−) were generated from fresh isolated BM cells grew in CellGro DC medium (CellGenix) containing 10% FCS (Pan Biotech), IL-4 (10 ng/ml) (R&D), and GM-CSF (10 ng/ml) (GIBCO). Medium was changed on days 2 and 4. On the seventh day, cells were analyzed by flow cytometry using the markers CD11c, F4/80, CD14, MHCII, CD11b, CD80, CD86, CD45, CD3, and CD19.
3
Priming Melan-A Specific CD8+ T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Naïve CD8+ T cells precursors for the Melan-A/MART-1 epitope ELA were primed in vitro using unfractionated PBMC protocol as described in [1] with minor modifications. Briefly, PBMC from HLA-A2+ healthy donors were thawed and seeded at 5 × 106 cells/ml in CellGro® DC medium (CellGenix™), supplemented with human GM-CSF (100 ng/ml; MACS Miltenyi Biotec) and IL-4 (20 ng/ml; Gemini Bio-products) in a 24-well tissue culture plate. After 24 h, Melan-A/MART-1 antigen (at 10 μg/ml) was added, in presence of M04 (final 50 μM) or 2'3'cGamp (5 μg/ml, Invitrogen) to induce maturation of resident dendritic cells. Twenty-four hours later and every 3 days, half of the media was replaced by fresh RPMI 1640 (Corning) supplemented with 8% human serum (Atlanta Biologicals) and IL-2 (20 U/ml, Miltenyi Biotech). On day 11, the CD8+ T cells frequency and were assessed by flow cytometry within the CD3+CD8+ T cell population using Melan-A –HLA-A2 tetramer staining after exclusion of dead cells.
4
Priming Naive CD8+ T Cells with Melan-A Antigen
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The priming assay is based on a previously described protocol68 (link). Briefly, DCs were generated from plastic-adherent human monocytes isolated from PBMCs of HLA-A2 + healthy donors. After 72 h of culture in GM-CSF/IL4-containing CellGro DC medium (CellGenix, cat. no. 20801-0500) with 1% human serum (HS), DCs were matured in medium containing 10 ng/mL IL4, 800 IU/mL GM-CSF, 10 ng/mL LPS, and 100 IU/mL IFNγ. Mature DCs were cultured in the presence or absence of 2.5 µg/mL Melan-A antigen peptide (ELAGIGILTV, JPT, cat. no. SP-MHCI-0006) for 16 h. The cells were then irradiated (30 Gy) and washed. Next, autologous naive CD8+ T cells were isolated from PBMCs with the EasySEP Human Naïve CD8+ T Cell Isolation Kit (Stem cell Technologies, cat. no. 19258) and co-incubated with mature DCs at a 4:1 ratio in CellGro DC medium with 5% HS and 30 ng/mL IL-21. Test antibodies at 10 µg/mL were added on day 0 and day 3 of co-culture. Fresh medium containing 5 ng/mL IL-7 and 5 ng/mL IL-15 was added on days 3 and 5. On day 10 of co-culture, T cells were harvested, counted, and stained with CD8, CD45RA, and CCR7 antibodies and ELAGIGILTV Dextramer (Immudex, cat. no. WB2162-APC) and analyzed by FACS.
5
Leukapheresis-Derived Monocyte Transduction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After informed consent, leukapheresis was obtained from healthy adult volunteers (HLA-A*02:01) with a COBE Spectra apheresis system. CD14+ monocytes were enriched by immunomagnetic separation using a GMP-compliant CliniMACS system (Miltenyi Biotec). Quantitative and qualitative analyses of the selected CD14+ fraction and flow through were performed by flow cytometry. From the enriched CD14+ fraction, 2 × 108 cells were resuspended in 25 ml of serum-free CellGro DC medium (CellGenix) and seeded in a 100 ml bag (CellGenix). Cells were preconditioned with 25 ml of medium containing hGM-CSF and human IL-4 cytokines (50 ng ml−1 each, CellGenix) for 8 h. Cells were transduced with 1 × 109 infective particles (multiplicity of infection of 5) in 50 ml medium containing Protamine sulfate (5 μg ml−1). The bag was incubated at 37 °C and 5% CO2 for 16 h. Next day, cells were washed three times with CellGro medium. After washing, cell number and viability was determined. Transduced cells were cryopreserved in aliquots of 2 × 106 cell ml−1 per vial. Surplus, non-transduced monocytes were cryopreserved in aliquots of 2 × 106 cell ml−1 per vial and 50 × 106 cell ml−1 per vial and were used as controls for the characterization experiments. Sterility tests were performed with the ‘Bactec' system (BD Biosciences).
6
Electroporation of mRNA into Expanded T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Expanded T cells were washed twice and resuspended in CellGro DC medium (CellGenix GmbH) and resuspended to 70 × 106 cells/mL. The mRNA was mixed with the cell suspension at 100 μg/mL, and electroporated in a 4-mm gap cuvette at 500 V and 2 ms using a BTX 830 Square Wave Electroporator (BTX Technologies Inc., Hawthorne, NY, USA). Immediately after transfection, T cells were transferred to complete culture medium at 37°C in 5% CO2 overnight to allow TCR expression.
7
Electroporation-based T Cell Transduction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expanded T cells were washed twice and resuspended in CellGro DC medium (CellGenix GmbH, Germany) at 7 × 107 cells/ml. 100 μg/ml mRNA encoding the TCR or sterile water for mock electroporations was mixed with the cell suspension, and electroporated in a 4-mm gap cuvette at 500 V and 2 ms using a BTX 830 Square Wave Electroporator (BTX Technologies Inc., USA). Immediately after transfection, the T cells were transferred to complete culture medium at 37 °C in 5% CO2 overnight to allow TCR expression. The T cells were then used fresh or frozen in aliquots.
8
Dendritic Cell and Activated T-Cell Generation
9
Autologous Tumor-Pulsed Dendritic Cell Vaccine
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mature DCs were achieved from ex vivo cultures of monocytes derived from peripheral blood mononuclear cells obtained by leukapheresis [15 (link),16 (link)]. Briefly, monocytes were enriched in immature DCs with CellGro DC medium (Cell Genix, Freiburg, Germany) added with Interleukin-4 (Cell Genix, Germany) 1000 IU/mL and GM-CSF (Cell Genix, Germany) 1000 IU/mL. On day 6, at least 90% of the culture was pulsed with 100 μg/mL of autologous tumor homogenate, whereas the remaining was pulsed with Immucothel (Biosyn Arzneimittel, Fellbach, Germany) 50 μg/mL as an immunization control. After overnight incubation and eliminating the previous culture medium, pulsed immature DCs were cultured for an additional 2 days with a cytokine maturation cocktail compound of Interleukin-6, Interleukin-1β, Tumor Necrosis Factor-α (Cell Genix, Germany), and ProstinE2 (Pfizer, Latina, Italy or Cayman, Ann Arbor, MI, USA). On day 9, 10 × 106 of DCs were harvested, washed, and resuspended in sterile saline for patient’s treatment (Figure 1 a). The remaining DC aliquots were frozen in autologous plasma and 10% dimethyl sulfoxide (Mylan, Dublin, Ireland) by automated freezing (Planer Ltd, Middlesex, UK) and stored in nitrogen vapor (Figure 1 b).
10
Phenotypic Characterization of NK Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GMP grade CellGro® DC medium was purchased from CellGenix (Freiburg, Germany). Fetal bovine serum (FBS) was from ExCell Biology (Shanghai, China), while penicillin, streptomycin, and glutamine were all from Gibco (Waltham, MA, USA). Interleukin-2 (IL-2) was from Peprotech (Rocky Hill, NJ, USA).
Fluorescent labeled anti-human monoclonal antibodies (mAb) used for flow cytometric analysis were CD3-PerCP, CD16-FITC, CD56-PE, CD56-FITC, NKp44-PE, NKp46-PE, NKG2D-PE, CD158a-FITC, CD158b-FITC, NKB1-FITC, CD107a-FITC and relevant isotype control antibodies. The first five antibodies were purchased from Miltenyi Biotec (Bergisch-Gladbach, Germany), and the others from BD Pharmingen (San Diego, CA). LEAFTM Purified anti-human CD16 antibody, purified anti-human CD274 (PD-L1), anti-human CD273 (PD-L2) and anti-human CD279 (PD1) antibodies were all purchased from Biolegend (San Diego, CA).
Fluorescent labeled anti-human monoclonal antibodies (mAb) used for flow cytometric analysis were CD3-PerCP, CD16-FITC, CD56-PE, CD56-FITC, NKp44-PE, NKp46-PE, NKG2D-PE, CD158a-FITC, CD158b-FITC, NKB1-FITC, CD107a-FITC and relevant isotype control antibodies. The first five antibodies were purchased from Miltenyi Biotec (Bergisch-Gladbach, Germany), and the others from BD Pharmingen (San Diego, CA). LEAFTM Purified anti-human CD16 antibody, purified anti-human CD274 (PD-L1), anti-human CD273 (PD-L2) and anti-human CD279 (PD1) antibodies were all purchased from Biolegend (San Diego, CA).
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!