Neutrophils were isolated from the peripheral blood of healthy volunteers, as described previously.25 (link) Briefly, heparinized human whole blood (25 mL) was mixed with 7 mL of 6% dextran solution (Wako, Osaka, Japan) and 10 mL of Hank’s balanced salt solution (HBSS)(-) (Gibco), and allowed to stand for 30 min at 25 °C until stratification had occurred. Leukocyte-rich plasma was collected and centrifuged at 500 × g in lymphocyte separation medium 1077 (Promo Cell, Heidelberg, Germany) for 30 min. A hypotonic (0.2%) saline solution was added to lyse the erythrocytes, and osmolality was restored by adding hypertonic (1.6%) saline. Neutrophils were adjusted to a final concentration of 1×107 cells/mL in HBSS. Neutrophil purity was determined using the Celltac MEK 6450 hematology analyzer (Nihon Kohden, Tokyo, Japan). Cell viability ≥95% was confirmed before each assay via trypan blue exclusion staining.
Lymphocyte separation medium 1077
Manufactured by PromoCell
Sourced in Germany
Lymphocyte Separation Medium 1077 is a sterile, isotonic density gradient medium designed for the isolation of lymphocytes from whole blood or other cell suspensions. The medium has a density of 1.077 g/ml and is optimized for the separation of mononuclear cells, including lymphocytes, from erythrocytes and granulocytes.
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Lab products found in correlation
Cd14 microbead, by Miltenyi Biotec (3 mentions)
Glutamine, by Lonza (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Sepmate 50 tube, by STEMCELL (1 mentions)
Cd14 magnetic microbeads, by Miltenyi Biotec (1 mentions)
Magnetic activated cell sorting (macs), by Miltenyi Biotec (1 mentions)
Cd45 microbeads, by Miltenyi Biotec (1 mentions)
Cell strainer, by Miltenyi Biotec (1 mentions)
Leucosep tube, by Greiner (1 mentions)
Anti cd14 microbeads, by Miltenyi Biotec (1 mentions)
Sepmate tube, by STEMCELL (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Fungizone, by Lonza (1 mentions)
Penicillin, by Lonza (1 mentions)
Streptomycin, by Lonza (1 mentions)
Celltrace cfse cell proliferation kit, by Thermo Fisher Scientific (1 mentions)
Human il 2 duoset elisa kit, by R&D Systems (1 mentions)
Human ifn γ duoset elisa kit, by R&D Systems (1 mentions)
22 protocols using lymphocyte separation medium 1077
1
Isolation of Neutrophils from Human Blood
2
Peripheral Blood Analysis in Neuroblastoma Patients
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Peripheral blood from patients with NB was collected from 35 stage IV NB patients pre and post-surgery between January 2021 and December 2021. Of the 35 patients, 24 were male and 11 were female, and the mean age at diagnosis was 26.11±9.13 months (range, 9–43 months). Gross total resection was achieved in 27 cases and subtotal resection in 8 cases. Peripheral blood was equally obtained from 10 healthy control patients (range, 22–32 months; mean age, 25.4±3.23 months) with benign pediatric surgical disease (oblique inguinal hernia) at the Children's Hospital of Fudan University (Shanghai, China) between January 2021 and December 2021.
Mononuclear cells were isolated using a Lymphocyte Separation Medium 1077 (PromoCell GmbH) from peripheral blood. Fresh or cryopreserved mononuclear cells were used for immunophenotype analysis. Plasma was used for the analysis of soluble NKG2D ligands.
The use of human material was approved (approval no. 2020238) by the Local Ethics Committee of Fudan University (Shanghai, China).
Mononuclear cells were isolated using a Lymphocyte Separation Medium 1077 (PromoCell GmbH) from peripheral blood. Fresh or cryopreserved mononuclear cells were used for immunophenotype analysis. Plasma was used for the analysis of soluble NKG2D ligands.
The use of human material was approved (approval no. 2020238) by the Local Ethics Committee of Fudan University (Shanghai, China).
3
Efficient Isolation of Peripheral Mononuclear Cells
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MNCs were collected by density gradient centrifugation as published before [46 (link)]; please see supplemental information. In short, the content of the leukocyte reduction chambers was diluted with phosphate-buffered saline (PBS, Sigma–Aldrich, St. Louis, MO, USA); 15 mL of Lymphocyte Separation Medium 1077 (Promocell, Heidelberg, Germany) was added to the lower chamber of SepMate-50 Tubes (Stemcell, Vancouver, Canada), and the diluted content of the leukocyte reduction chambers was added on top of the inner barrier. The special insert of these tubes allows density gradient centrifugation at 1200 g for 10 min with full break, significantly reducing the processing time of MNC isolation. The buffy coat was washed once with PBS and resuspended at 2 × 107 cells/mL in full MNC medium [RPMI1640 (Sigma–Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS Gold; Seraglob, Switzerland), glutamine, and antibiotics (both Lonza, Basel, Switzerland)].
4
Isolation and Culture of Macrophages
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Macrophages were isolated from ascites (TAMs) or peritoneal lavage fluids (pMPHs) by density gradient centrifugation (Lymphocyte Separation Medium 1077; PromoCell) and subsequent enrichment on magnetic CD14 microbeads (Miltenyi Biotech). Tumor cells and CD3+ T cells were isolated as described [38 (link)]. MDMs were generated from monocytes (6-day differentiation for RNA experiments, 10-day cultures for flow cytometry) from healthy donors as described [75 (link)] and in RPMI with human AB serum.
5
Isolating Ascites Tumor Cells and Spheroids
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Mononuclear cells were isolated from ascites by Lymphocyte Separation Medium 1077 (PromoCell) density gradient centrifugation. Tumor spheroids were separated by filtration using 30 μm and 40 μm cell strainer (Miltenyi Biotech) resulting in either spheroids of medium size (30–40 μm = “m”) or large size (>40 μm = “L”). Small tumor spheroids (<30 μm = “s”) and tumor single cells (sc) were further purified by depletion of peritoneal leucocytes using CD45 microbeads and magnetic cell sorting (MACS) (Miltenyi Biotech). Purified tumor cells were lysed in PeqGold (Peqlab) for RNA preparation, analyzed by flow cytometry, or cultured for testing of chemoresistance. The purity of tumor spheroids/cells was >90 % EpCAM+ cells, except for sample OC84s (>85 %, Additional file 4 : Table S2).
6
Isolation and Analysis of Tumor-Associated Macrophages
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Mononuclear cells were isolated from ascites by Lymphocyte Separation Medium 1077 (PromoCell) density gradient centrifugation and further purified by magnetic cell sorting (MACS) using CD14 microbeads (Miltenyi Biotech) [16 (link)]. TAMs were analyzed by flow cytometry for surface expression of CD14, CD16, CD32, CD64, CD163 and CD206 as described [16 (link)]. Tumor cell spheroids and T cells were analyzed as previously published [21 (link)].
7
Isolation and culture of DBA/2-specific T cells
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DBA/2-specific T cells were obtained from recipient mouse lines (WT, GzmB KI, Bonzo or CD8-τGFP). Briefly, splenocytes were isolated from 8 to 20 week-old mice, as described before [26 (link)]. Splenocytes were incubated with crude DBA/2 lysate (~1.5 mg/mL total protein concentration) in RMPI culture medium containing 50 µM β-mercaptoethanol, 10% fetal calf serum, 1% Penicillin/Streptomycin and supplemented with 50 U/mL IL-2 for two days. Blast cells were generated after two days in culture. 100 U/mL IL-2 was added to the culture medium at day 3 to induce proliferation of T cells. T cells were separated from mixed splenocytes at day 3 or 4 using Lymphocyte Separation Medium 1077 (PromoCell, Heidelberg, Germany) with a gradient centrifugation at 400× g, 30 min at room temperature. For T cell killing and migration experiments, day 4 and 5 effector T cells were used for co-culture with islet cells after lymphocyte purification.
8
Isolation of Tumor Cell Spheroids and Immune Cells
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Tumour cell spheroids, TAM and TAT were isolated from ascites essentially as described.17 , 25 Briefly, mononuclear cells were isolated from ascites by density gradient centrifugation (Lymphocyte Separation Medium 1077; PromoCell). Medium size (‘M’) and large (‘L’) tumour cell spheroids were obtained by filtration using 30 and 100 μm cell strainer (Miltenyi Biotech). Smaller tumour spheroids (<30 μm; ‘s’) and single tumour cells (sc) were further enriched by depletion of CD45+ leukocytes by magnetic cell sorting (MACS; Miltenyi Biotech, Bergisch Gladbach, Germany). TAM were purified by selection for CD14+ cells on MACS microbeads. TAT were isolated from ascites as CD3+ cells by MACS. All microbeads for MACS (CD3, CD14, CD45) were obtained from Miltenyi Biotech. Cell populations with a purity of > 95%, as determined by flow cytometry, were used for subsequent analysis.
9
Isolation and Analysis of Tumor-Associated Macrophages
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Mononuclear cells were isolated from ascites by Lymphocyte Separation Medium 1077 (PromoCell) density gradient centrifugation and further purified by magnetic cell sorting (MACS) using CD14 microbeads (Miltenyi Biotech). TAMs were directly analyzed by FACS as described below or lysed in PeqGold (Peqlab) for RNA preparation.
10
Isolation of Mouse Lymphocytes
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Mouse peripheral blood, splenic, and thymic lymphocytes were isolated using Lymphocyte Separation Medium 1077 (PromoCell, Heidelberg, Germany). Briefly, peripheral blood and cell suspensions were obtained by crushing the spleen and thymus. Suspensions were diluted with an equal volume of sterile PBS and the diluted cell suspension was carefully overlaid on three volumes of Lymphocyte Separation Medium 1077 in 15 ml tubes and centrifuged at 400 × g for 40 min without brakes. Mouse lymphocytes were removed from the liquid/medium interface and washed three times with 0.1% bovine serum albumin (BSA) in PBS.
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