The methods of maintaining RAW264.7 cells and further differentiating them into osteoclasts were descripted in our previous publication [15] . In brief, RAW264.7 cells were seeded at 5 Â 10 3 cells/well in a 96-well plate, and cultured with 50 ng/ml soluble murine RANKL plus IL-26 or IgG control for 5 days. Human peripheral blood mononuclear cells (PBMCs) were obtained from healthy adult volunteers, with consent and ethical approval (Tri-Service General Hospital, 1-1-4-05-128), and isolated by density centrifugation using lymphocyte separation medium according to the manufacturer's instructions (Lonza, Walkersville, MD, USA). Moreover, PBMCs were resuspended in alpha minimal essential medium (a-MEM) (Gibco BRL/Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 30 ng/ml macrophage colony-stimulating factor (M-CSF), penicillin (100 U/ ml) and streptomycin (100 mg/ml), and plated in 96-well plates at a density of 4 Â 10 5 cells/well for osteoclast differentiation as previously described [16] . All differentiation media were replaced on days 23.
Lymphocyte separation medium
Manufactured by Lonza
Sourced in United States, Switzerland, Germany
Lymphocyte separation medium is a laboratory reagent used to isolate and enrich lymphocytes from whole blood samples. It is a density gradient solution that allows the separation of different blood cell types based on their density differences when centrifuged. This product facilitates the study and analysis of lymphocytes, which are an important component of the immune system.
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Lab products found in correlation
Easysep human na ve cd4 t cell enrichment kit, by STEMCELL (3 mentions)
Anti cd3 anti cd28 coated beads, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Rnaprotect, by Qiagen (2 mentions)
Monocyte isolation kit 2, by Miltenyi Biotec (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
Biotarget medium, by Sartorius (1 mentions)
Rpmi medium, by Thermo Fisher Scientific (1 mentions)
Mm 1s, by American Type Culture Collection (1 mentions)
Fungizone, by Thermo Fisher Scientific (1 mentions)
L glutamate, by Thermo Fisher Scientific (1 mentions)
6 well plate, by Corning (1 mentions)
Gm csf, by R&D Systems (1 mentions)
Interleukin 4 (il 4), by R&D Systems (1 mentions)
Human serum, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
42 protocols using lymphocyte separation medium
1
Osteoclast Differentiation from RAW264.7 and PBMCs
2
Isolation of Lung Mononuclear Cells
3
PBMC Isolation and Culture for Plasma Cell Malignancies
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Peripheral blood mononuclear cells (PBMC) were from healthy donors obtained from the Israeli Blood Bank (Tel-Hashomer, Israel), the Pheresis Collection Unit, (IRB-approval #0458-19-HMO) or from blood of patients with plasma cell malignancies (IRB-approval #0253-20-HMO). BCMA+ cell lines are RPMI8226(ATCC/CCL-155), (NCI)-H929(ATCC/CRL-9068) and MM1.S(ATCC/CRL-2974). K562(ATCC/CCL-243) is an erythroleukemia line BCMAneg. K562-BCMA was engineered to overexpress BCMA. Cell lines were cultured in RPMI medium (Invitrogen, Carlsbad, CA), with 10% heat-inactivated fetal bovine serum (Biological Industries, Israel), at 37°C and 5% CO2. Bone marrow (BM) aspirates from patients with plasma cell dyscrasias were obtained in accordance with Helsinki approval from the Ethical Committee of Hadassah Ein-Kerem Medical Center. BM-derived mononuclear cells were isolated by centrifugation over a density gradient medium (lymphocyte separation medium, Lonza). Lymphocytes were cultured in BioTarget medium (Biological Industries, Israel) with 10% fetal bovine serum, 1% L-glutamine, 1% penicilin/streptomycin and 300 IU/mL IL-2 (Peprotech-Asia, Israel).24-26
4
Plasma and Lymphocyte Isolation
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Each 5 mL blood sample was divided in two: 2 mL was centrifuged for 5 minutes at 12,000 rpm for plasma isolation, and 3 mL was used for lymphocyte isolation using Lymphocyte Separation Medium (Lonza Walkersville Inc., USA) following supplier´s indications.
5
Differentiation of Monocytes into Dendritic Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation of heparinized blood using Lymphocyte Separation medium (Lonza, Walkersville, MD, USA). PBMCs were cultured in RPMI-1640 (Invitrogen, San Giuliano Milanese, Italy) in 6-well plates (Corning, Euroclone, Milano, Italy) at a density of 2.5 × 106/mL at 37 °C in a 5% CO2 humidified atmosphere. After l h, non-adherent cells were removed. Adherent cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) (GIBCO, Thermo Fisher, Milano, Italy), 1% L-glutamate (ICN), 100 U/mL streptomycin or penicillin, and 80 µL fungizone (GIBCO, Thermo Fisher, Milano, Italy). To obtain DCs, 1000 U/mL of granulocyte-monocyte colony stimulating factor (GM-CSF, RD System, Minneapolis, Minnesota, USA) and 800 U/mL of interleukin 4 (IL-4, RD System, Minneapolis, Minnesota, USA) were added to the culture medium on Day zero and Day 3. Monocytes differentiated into immature DCs during 6 days of culture. DCs were harvested at Day 6 or 7.
6
PBMC Isolation and Cryopreservation
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Peripheral blood was collected into BD Vacutainer® CPTTM Cell Preparation Tube with Sodium Citrate (BD Biosciences, Franklin Lakes, NJ, USA). PBMC were isolated from whole blood using SepMate 50-ml tubes with a high-density polyethylene membrane (Greiner Bio-One, Frickenhausen, Germany) and lymphocyte separation medium (Lonza, Lysaker, Norway), centrifuged for 10 min at 1,200 g. Cells were cryopreserved and frozen down in RPMI 1640 medium (Invitrogen), 25% v/v human serum (Sigma-Aldrich), 7% v/v DMSO in liquid nitrogen.
7
Evaluating FPTM Effects on T-cell Activation
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Lymphocyte separation medium (Lonza) was used to purify lymphocytes from fresh human blood obtained from healthy donors using density gradient centrifugation. (The use of human tissue samples was approved by the Franciscan University of Steubenville's Institutional Review Board.) The lymphocytes were plated in 96‐well plates at a concentration of 500,000 cells/ml with a total volume of 200 μl per well. To test the FPTM, some of the wells were supplemented with 20% (40 μl) of FPTM. (20% FPTM was used as it was the lowest concentration of FPTM tested that displayed a consistent ability to suppress INF‐γ secretion across lots.) Five different vials of FPTM were tested, each from different lots (A–E), and all vials were tested in duplicate.
The T cells were activated by adding 2uls of CD3/CD28 T‐activator Dynabeads™ to each well per the manufacturer's recommendations. The negative control well did not receive beads while the positive control well did not receive FPTM. The cells were incubated in the presence of the Dynabeads™ for 48 h. After 48 h, the contents of each well were transferred to Eppendorf tubes and spun in a microcentrifuge at maximum speed for 5 min. The supernatant was removed and frozen at −80°C for further analysis. After thawing, the level of INF‐γ in the supernatant was then measured using an INF‐γ ELISA test manufactured by RayBiotech.
The T cells were activated by adding 2uls of CD3/CD28 T‐activator Dynabeads™ to each well per the manufacturer's recommendations. The negative control well did not receive beads while the positive control well did not receive FPTM. The cells were incubated in the presence of the Dynabeads™ for 48 h. After 48 h, the contents of each well were transferred to Eppendorf tubes and spun in a microcentrifuge at maximum speed for 5 min. The supernatant was removed and frozen at −80°C for further analysis. After thawing, the level of INF‐γ in the supernatant was then measured using an INF‐γ ELISA test manufactured by RayBiotech.
8
Isolation and Purification of CD4+ T Cells
9
Isolation of T cells from Childhood T-ALL Patients
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Bone marrow samples from childhood T-ALL patients at diagnosis, collected at the Hematology Laboratory of Peking University First Hospital between 2010 and 2015, were retrospectively studied. The current study was approved by the Biomedical Ethical Committee of Peking University. Mononuclear cells from patients' bone marrow were separated using the lymphocyte separation medium following the manufacturer's instructions (Lonza Walkersville Inc.). T cells were isolated from PBMC by immunomagnetic negative selection using EasySep™ Human T Cell Isolation Kit (StemCell Technologies Inc.). The purity of the isolated T cells was >95%, as determined by flow cytometric analysis. T cells from 5 healthy donors were pooled and used as an internal control [32 (link)].
10
Monocyte and MDSC Population Dynamics in Melanoma Immunotherapy
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Blood was collected at treatment cycles 1 and 4, immediately prior to treatment (Day 1 for Cohorts A and B, or Day 0 for Cohort C) and 2 days following vaccination (Day 3), to analyze the potential systemic effects of Melanoma GVAX on circulating monocyte populations. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation (Lymphocyte Separation Medium, Lonza, Walkersville, MD, USA) and cryopreserved. All specimens from each patient were thawed and analyzed simultaneously. To assess effects on the numbers and activation state of circulating monocytes, PBMCs were stained with anti-HLA-DR, -CD14, -CD11b, or isotype-matched control mAbs (BD Biosciences, San Jose, CA, USA), and were analyzed by flow cytometry. The mean fluorescence intensity (MFI) of HLA-DR expression on CD14+CD11b+ events was analyzed as an indicator of monocyte activation. MDSCs were defined as FSChiSSChiCD14+CD11b+HLA-DRlo/(−), where the region of HLA-DRlo monocytes was defined relative to gating on HLA-DR(−) lymphocytes. MDSCs were quantified as a percentage of CD14+CD11b+ monocytes, or by absolute number per μl of whole blood. Data were acquired on the BD FACSCalibur and analyzed using Flow Jo software (TreeStar, Ashland, OR, USA).
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