Periphery blood mononuclear cells (PBMCs) were isolated using a lyse-then-wash step as described previously [25 (link)]. Basically, a total of 20 ml venous blood was collected directly into a heparinized tube and diluted with sterile pH 7.2 phosphate-buffered saline (PBS) at room temperature. The PBMCs were then isolated by density gradient centrifugation using Ficoll-Paque Plus (MP Biomedicals, Santa Ana, CA, USA). The acquired PBMCs were washed twice with PBS and resuspended at 1 × 107 cells/ml for culture.
Ficoll paque plus
Manufactured by MP Biomedicals
Sourced in United States
Ficoll-Paque Plus is a density gradient medium used for the separation and purification of cells and cell fractions. It is a sterile, pyrogen-free solution composed of sucrose and epichlorohydrin-cross-linked Ficoll.
Automatically generated - may contain errors
Lab products found in correlation
Rhil 10, by Thermo Fisher Scientific (1 mentions)
Anti il 10 monoclonal antibody, by R&D Systems (1 mentions)
Cd4 microbeads, by Miltenyi Biotec (1 mentions)
Rhil 25, by Thermo Fisher Scientific (1 mentions)
Recombinant human il 2, by Thermo Fisher Scientific (1 mentions)
Rhil 33, by Thermo Fisher Scientific (1 mentions)
4 protocols using ficoll paque plus
1
PBMC Isolation from Venous Blood
2
Modulation of Th2 Responses by mDC-derived sEVs
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Human PBMCs from patients with AR or buffy coat from “anonymous donors” were isolated by density centrifugation with Ficoll‐Paque Plus (MP Biomedicals, Santa Ana, CA, USA). CD4+ T cells were sorted from PBMCs of buffy coat of human volunteers using the CD4 Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). sEV‐mDCs were harvested and washed twice, and then were co‐cultured with allogeneic PBMCs isolated from patients with AR or freshly sorted CD4+ T cells (1:10) for 5 days in RPMI 1640 (Hyclone, Pittsburgh, PA, USA) supplemented with 10% FBS and IL‐2 (100 U/mL). Cell counting was performed manually using a hemocytometer and the cell viability was over 95%. In some experiments, CD4+ T cells were treated with the rhIL‐10 (10 ng/mL; PeproTech Inc.) together with mDC, or incubated with anti‐IL‐10 monoclonal antibody (75 ng/mL) or IL‐10Rα blocking antibody (5 μg/mL; both from R&D systems, Minneapolis, MN, USA) for 30 min before the co‐culture. The PBMCs or the T cells were collected for the analysis of intracellular Th2 cytokines using flow cytometry, and the supernatants were used for the evaluation of IL‐13, IL‐9, IL‐10, or IFN‐γ. T cells were also evaluated for IL‐10+Foxp3+ T cells or Treg cells.
3
Isolation of PBMCs from Whole Blood
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Peripheral blood mononuclear cells (PBMCs) were separated from whole human blood using gradient centrifugation by Ficoll-Paque Plus (MP Biomedicals, Illkirch, France) as reported by Lohr et al. [31 (link)]. Briefly, whole blood sample was 5 times diluted using freshly prepared PBS and then overlaid dropwise on Ficoll. The monolayer of PBMCs was separated using gradient centrifugation at 2000 rpm for 30 min. The recovered cells were collected and washed 3 times using PBS.
4
Analyzing ILC2 Functions in Allergic Rhinitis
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The study was approved by the Ethics Committee of The First Affiliated Hospital, Sun Yat‐sen University. Written informed consents were obtained from all participants. PBMCs were isolated from healthy controls (n = 6) and AR patients (n = 8) by density centrifugation with Ficoll‐Paque Plus (MP Biomedicals, Santa Ana, California). Control subjects did not report any symptoms of AR and did not have a history of allergic airway inflammation. The inclusion criteria for patients with AR were established according to the Initiative on Allergic Rhinitis and its Impact on Asthma (ARIA): (a) history of nasal symptoms by nose itching, obstruction, sneezing, and rhinorrhea; (b) positive specific immunoglobulin E (sIgE). Patient information can be found in Table S1 .
The isolated PBMCs were cocultured with iPSC‐MSCs in a ratio of 10:1 in RPMI 1640 supplemented with 10% FBS, 1% penicillin‐streptomycin. Recombinant human (rh)IL‐2 (20 U/mL), rhIL‐25 (10 ng/mL), and rhIL‐33 (10 ng/mL; PeproTech, Inc, Rocky Hill, New Jersey) were added into the coculture system as a positive control following the previous study.16 (link) ELISA, quantitative PCR and flow cytometry were performed to analyze the ILC2 functions from the coculture system after 3 days.
The isolated PBMCs were cocultured with iPSC‐MSCs in a ratio of 10:1 in RPMI 1640 supplemented with 10% FBS, 1% penicillin‐streptomycin. Recombinant human (rh)IL‐2 (20 U/mL), rhIL‐25 (10 ng/mL), and rhIL‐33 (10 ng/mL; PeproTech, Inc, Rocky Hill, New Jersey) were added into the coculture system as a positive control following the previous study.16 (link) ELISA, quantitative PCR and flow cytometry were performed to analyze the ILC2 functions from the coculture system after 3 days.
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