Prior to the first radiotherapy treatment, 3 mL of heparinized whole peripheral blood was collected from each of the patients. Lymphocytes were freshly isolated through Ficoll-Paque density gradient (Biochrom, Berlin, Germany) centrifugation for 15 minutes at 1200 G and 24°C. Subsequently, the mononuclear cell layer was harvested and these cells were washed by resuspension with RPMI 1640 medium (Sigma-Aldrich, Taufkirchen, Germany) and centrifugation for 10 minutes at 300 g and 24°C. This last step was repeated once. The washed cells were added to a nutrient medium consisting of 7.2 mL RPMI 1640 medium, 1.8 mL fetal bovine serum (Biochrom), 100 μL Penicillin (10.000 U/mL), and Streptomycin (10.000 μg/mL) (Gibco, Life Technologies, Darmstadt, Germany). The cell suspension was divided into ten 1.4 mL U-bottom push cap tubes (Micronic, Wernberg-Köblitz, Germany) with 400 μL each.
Ficoll paque density gradient
Manufactured by Harvard Bioscience
Sourced in Germany
Ficoll-Paque density gradient is a laboratory tool used for the separation and purification of cells, organelles, and other biological particles based on their density differences. It is a sterile, pyrogen-free, and endotoxin-tested solution that can be used in a variety of applications, such as the isolation of mononuclear cells from blood samples. The product's core function is to facilitate the separation of different cellular components through density gradient centrifugation.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (1 mentions)
Rpmi 1640 medium, by Harvard Bioscience (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Human serum, by Merck Group (1 mentions)
Cd34 microbead kit ultrapure human, by Miltenyi Biotec (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Interleukin 7 (il 7), by Miltenyi Biotec (1 mentions)
Egfr pe, by BioLegend (1 mentions)
Egfr fitc 13 egfr ruo, by BD (1 mentions)
Pd1 pe pd, by Miltenyi Biotec (1 mentions)
Lag3 apc, by BioLegend (1 mentions)
Texmacs media, by Miltenyi Biotec (1 mentions)
Cd4 and cd8 microbeads, by Miltenyi Biotec (1 mentions)
Il 15, by Miltenyi Biotec (1 mentions)
3 protocols using ficoll paque density gradient
1
Lymphocyte Isolation for Radiotherapy
2
CD34+ HSC Isolation and Cryopreservation
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Mononuclear cells were isolated from blood products by Ficoll‐Paque density gradient centrifugation (Biocoll; Biochrom, Germany). CD34+ HSC were isolated by positive magnetic selection (human CD34 MicroBead Kit UltraPure; Miltenyi Biotec, Germany) according to the manufacturer's instructions. Cryopreservation was performed in human serum supplemented with 10% dimethyl sulfoxide (both from Sigma‐Aldrich, Germany) at a cooling rate of 1°C/min.
3
Engineered CAR T Cell Generation
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PBMC were isolated from whole peripheral blood, acquired from healthy volunteer donors at the University Children’s Hospital Tuebingen, by Ficoll-Paque density gradient centrifugation (Biocoll, Biochrom). T cells were sequentially isolated using CD4 and CD8 microbeads (Miltenyi Biotec) and mixed at a 1:1 ratio. T cells were activated with TransActTM (anti-CD3 and anti-CD28 agonistic signal, Miltenyi Biotec) and cultivated in TexMACS media (Miltenyi Biotec) supplemented with 10 ng/mL IL7 and 5 ng/mL IL15 (Miltenyi Biotec). After 24 h, activated T cells were transduced at a multiplicity of infection (MOI) of 3. Transduced T cells were maintained at 0.5–2 × 106 cells/mL in IL7/IL15 containing TexMACS® media. On day +7, CAR transduction efficiency and CD4/CD8 ratio were determined by flow cytometry using the antibodies: CD4-BUV395 (SK3, BD Bioscience), CD8-APC (BW135/80, Miltenyi Biotec) and EGFR-PE (AY13, BioLegend). Tonic signaling was assessed by expression of the exhaustion markers PD-1, TIM-3, and LAG-3 via flow cytometry on day +21 of culturing using the antibodies: EGFR-FITC (13/EGFR (RUO), BD Bioscience); PD-1-PE (PD1.3.1.3, Miltenyi); TIM-3-PE-/Dazzle594 (F38-2E2, BioLegend); LAG-3-APC (7H2C65, BioLegend) and LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (ThermoFisher).
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