Peripheral blood (buffy coat) was obtained from healthy blood donor drawn at blood Transfusion Center (Azienda Ospedaliera di Perugia) in accordance with the written approval of the Director of the Blood Transfusion Service. Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation using Ficoll-Paque medium (#17-1400-02, GE Healthcare) from buffy coat diluted 1:4 with PBS 1X. Dendritic cell was enriched from PBMCs by immunomagnetic depletion of monocytes and macrophages (CD14+ CD16+), B cells (CD19+), T cell (CD3+), granulocytes (CD66b+), neutrophils (CD16+) and NK cells (CD335+). Cells were resuspended in RPMI 1640 (#11875093, Thermo Fisher) supplemented with 0.1 mM Non-essential Amminoacids (#11140-035 Thermo Fisher), 1 mM Sodium Pyruvate (#11360-070, Thermo Fisher), 5 mM glutamine (#25030-024, Thermo Fisher), 50 μM 2-Mercaptoethanol (#31350-010, Thermo Fisher), 100 U/ml penicillin, 100 g/ml streptomycin (#15140-122, Thermo Fisher) and 10% FBS (#10270-106, Thermo Fisher) (complete RPMI). cDC1 and cDC2 were sorted into complete RPMI by FACSAria Fusion as CD141+ Cleac9+ HLA-DR+CD11c–CD3–CD14–CD16–CD19–CD335–CD66b–(cDC1) and CD1c+ HLA-DR+CD11c+CD3–CD14–CD16–CD19–CD335–CD66b– (cDC2). Sort purity of > 95% was confirmed by post-sort analysis before cells were used for further experiments.
Ficoll paque medium
Manufactured by GE Healthcare
Sourced in United States, Sweden
Ficoll-Paque medium is a density gradient medium used for the isolation and purification of cells and other biological particles. It is composed of a mixture of sucrose and Ficoll polymer. The medium allows for the separation of different cell types based on their density during centrifugation.
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Lab products found in correlation
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Rt pcr kit, by Takara Bio (2 mentions)
Thermal cycler dice real time system, by Takara Bio (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Rosettesep human mesenchymal stem cell enrichment cocktail, by STEMCELL (1 mentions)
40 μm cell strainer, by BD (1 mentions)
Percoll, by GE Healthcare (1 mentions)
Moflo astrios cell sorter, by Beckman Coulter (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
35 mm glass bottom dishes, by MatTek (1 mentions)
Easysep human cd4 t cell isolation kit, by STEMCELL (1 mentions)
Leucosep tube, by Greiner (1 mentions)
Celltiter blue cell viability assay, by Promega (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
10 protocols using ficoll paque medium
1
Isolation and Purification of Human cDC1 and cDC2
2
PBMC Gene Expression Analysis Protocol
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Gene expression was analyzed using peripheral blood mononuclear cells (PBMCs). Blood samples were collected in ethylenediaminetetraacetic acid (EDTA) tubes. PBMCs were isolated by density gradient centrifugation at 3000 rpm for 30 min in Ficoll–Paque medium (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Total RNA was extracted using the TRI reagent, and cDNA was synthesized with 1 μg of total RNA using an RT-PCR Kit (Takara Bio Inc., Shiga, Japan). PCR was performed using a Thermal Cycler Dice Real-Time System (Takara Bio Inc., Shiga, Japan). Amplification was performed under the following conditions: 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 10 s, and extension at 72 °C for 10 s. Relative gene expression was analyzed using the comparative Ct method. Gene expression was determined using the 2−ΔΔCt method as the fold difference between ΔCt of the target sample and ΔCt of the calibrator sample. All reactions were performed in triplicates.
3
Circadian Gene Expression Analysis in PBMCs
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Gene expression analysis was performed on peripheral blood mononuclear cells (PBMCs). Blood samples were collected in ethylenediaminetetraacetic acid tubes. PBMCs were isolated via density gradient centrifugation at 3000 rpm for 30 min in Ficoll–Paque medium (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Total RNA was extracted using TRI reagent, and cDNA synthesis was carried out with 1 μg of total RNA using an RT-PCR Kit (Takara Bio Inc., Shiga, Japan). The qPCR primers for CLOCK, BMAL1, PER1, PER2, PER3, CRY1, CRY2, REV-ERBα, and REV-ERBβ were designed. Polymerase chain reaction was carried out using a Thermal Cycler Dice Real-time System (Takara Bio Inc.) and gene-specific primers designed from sequences obtained from the NCBI nucleotide sequence database (Supplementary Table S1 ). PCR was conducted using a Thermal Cycler Dice Real Time System (Takara Bio Inc.) with the following conditions: 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 10 s, and extension at 72 °C for 10 s. Relative gene expression was determined using the comparative Ct method, and gene expression was calculated using the 2−ΔΔCt method to ascertain the fold difference between ΔCt of the target sample and ΔCt of the calibrator sample. All reactions were performed in triplicate.
4
Isolation of Bone Marrow-Derived Mononuclear Cells
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Bone marrow samples were aspirated from the iliac crest of healthy donors (60 ml, n = 21, median age 25, range 18–45). All participants in the study gave a written informed consent. The procedure was approved by the Swedish local ethics committee in Lund (Regionala Etikprövningsnämden Lund, EPN, protocol Dnr2009/532) and all experimental protocols were performed according to EPN’s guidelines. Bone marrow mononuclear cells (BM-MNCs) were isolated by density gradient centrifugation (Ficoll-paque medium, GE Healthcare Life Sciences) combined with prior incubation with RosetteSep Human Mesenchymal Stem Cell Enrichment Cocktail (StemCell Technologies) for lineage depletion (CD3, CD14, CD19, CD38, CD66b and glycophorin A).
5
Isolation and Purification of Tumor-Infiltrating Lymphocytes
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TILs were prepared as described previously, with some modifications [47 (link)]. Briefly, mice were challenged with TC-1 tumor cells and immunized with PEK protein vaccine and/or anti-PD-L1 Ab. The mice were sacrificed and tumors excised 7 days after the last protein immunization (on day 28 after tumor challenge) (Figure 5 A). The tumors were dissected into small fragments and digested in 0.1 mg/mL collagenase in CTL medium at 37 °C overnight. After filtering through a 40 μm cell strainer (BD Falcon), the cell suspension was incubated for 30 min at 37 °C. After washing with CTL medium, mixing cell suspensions of CTL medium and balanced salt medium were layered on Ficoll–Paque medium (GE Healthcare, Pittsburgh, PA, USA) before centrifugation. TILs from the white interface layer were collected and washed with PBS.
6
PBMC Isolation from Healthy Donors
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Fresh whole blood was collected from healthy male volunteers aged 20–35 years presented in Tartu University Hospital Blood Bank. Written informed consent was obtained from study participants. Participants had to state that they had not used narcotics within the previous year. Human PBMCs were isolated by density centrifugation on the day of blood collection using Ficoll-Paque medium (GE Healthcare, USA) according to manufacturer’s instructions. PBMCs were stored at − 150 °C until use.
7
Isolation of Lymphocytes from Blood and Tissue
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Peripheral blood mononuclear cells (PBMC) were isolated from EDTA-K2 anticoagulated blood by Ficoll-Paque Medium (GE Healthcare, Uppsala, Sweden) using density gradient-centrifugation. The fresh tumor samples or normal lung tissues were manually cut into 3–5 mm2 fragments, ground and filtered through a 70 µm strainer, followed by centrifugation over a two-step gradient of 40% and 70% Percoll (GE Healthcare, Uppsala, Sweden). Lymphocytes were collected between the two layers. Lymph nodes were gently minced through 70 µm strainer and collected in sterile tube containing culture medium.
8
PBMC Stimulation and Supernatant Collection
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Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA-K2 anticoagulated blood using Ficoll-Paque Medium (GE Healthcare, Uppsala, Sweden) and density gradient centrifugation. The PBMCs (2 × 106 cells/well, 6-well plate) were cultured alone or treated with 1 µg/mL CD3 monoclonal antibody (mAb) (BD Bioscience, San Jose, CA, USA) in RPMI 1640 medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. Two days later, the supernatants were collected and used for co-culture with lung cancer cells.
9
Isolation and Characterization of Treg Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood samples by density gradient centrifugation using Ficoll-Paque medium (GE Healthcare, Chicago, IL, USA; Supplement Material 2 ). The following antibodies were used for characterization of the Treg cell pool in purified PBMCs incubated within 15 min: anti-CD4-FITC (A07750), anti-CD25-PC5 (IM2646), and CD127-PE (IM1980U; all from Beckman Coulter Inc., Brea, CA, USA). Labeled Treg cells were isolated through fluorescence-activated cell sorting (FACS) using the MoFlo Astrios Cell Sorter (Beckman Coulter).
10
Isolation and Characterization of Human CD4+ T Cells
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