Ficoll paque plus

Manufactured by Cytiva

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Ficoll-Paque Plus is a laboratory product used for the separation and isolation of cells from biological samples. It is a sterile, pyrogen-tested medium composed of sucrose and sodium diatrizoate. Ficoll-Paque Plus has a defined density that allows the separation of different cell types based on their density when centrifuged.

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Ficoll-Paque (225 citations) Ficoll (53 citations) Ficoll-Paque Plus solution (21 citations) Ficoll-Paque Plus gradient (8 citations) Ficoll Paque™ Plus 206 (8 citations) Ficoll-Paque PLUS density gradient medium (8 citations) Ficoll-Paque PLUS density gradient (8 citations) Ficoll-Paque PLUS Media (7 citations) Ficoll-Paque PLUS density (2 citations) Ficoll-Paque™ PLUS protocol (2 citations)

419 protocols using ficoll paque plus

Isolation and Characterization of hBM-MSCs

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In order to prepare hBM-MSCs, bone marrow aspirate was harvested from the iliac crest of a normal donor who was negative for HIV, HBV and HCV laboratory tests, after informed consent was obtained. Then, hBM-MSCs were cultured and isolated as previously reported.^{26 (link)} Mononuclear cells were isolated by gradient centrifugation at 2500 rpm for 30 minutes on Ficoll-Paque^TM Plus (Amersham Pharmacia Biotech, Uppsala, Sweden). Mononuclear cells were then plated at a concentration of 10-30 × 10³ cells/cm² in Dulbecco’s Modified Eagle Medium (DMEM) containing 20% (v/v) of FBS. Non-adherent cells were removed 2 days later, and fresh medium was added. When the cultures reached 80-100% confluence, hBM-MSCs were trypsinized and subcultured. The purity of hBM-MSC suspensions was evaluated by flow cytometry using the following monoclonal antibodies: CD105-PE, CD45-FITC and CD90-PerCP, along with corresponding isotype controls (BD Biosciences, San Jose, CA, USA). Finally, the suspensions were analyzed utilizing a BD FACS Canto^TM flow cytometry System (BD Biosciences, CA).

Mahmoudi T., Jalili A., Abdolmohammadi K., Fakhari S., Pahlavan F., Shekari A., Nikkhoo B., Tayebi L, & Rahmani M.R. (2021). Human Bone Marrow Mesenchymal Stromal Cells Attenuate Tissue Injury and Reduce Inflammation in Experimental Acute Pancreatitis. Advanced Pharmaceutical Bulletin, 12(2), 375-382.

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In-House HCV RT-PCR Validation

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To validate practical application of developed in-house HCV real-time RT-PCR, the presence of HCV RNA was determined in both sero-positive and -negative plasma specimens collected from 38 patients with definitive diagnosis of HCV infection and 14 normal healthy donors, respectively. Patients were already known to have positive results for HCV antibody and RNA. They all were new cases and had received no treatment on the time of sample collection. To assess whether the developed real time RT-PCR would be able to detect the HCV genotypes 1 to 4 which are dominant in the region, HCV genotypes were determined for all the plasma samples using a commercially available kit (Sacace Biotechnologies Srl, Caserta, Italy). There were 17 genotype 1, 2 genotype 2, 17 genotype 3 and 2 genotype 4. All the blood samples were identified as HIV-1 antibody negative by anonymous testing with ELISA method (anti-HIV Tetra ELISA, Biotest Co. Germany). Blood samples were centrifuged at 805 × g for 10 minutes and the plasma was recovered. PBMCs were isolated from the remaining volume by 3:1 dilution with phosphate buffer saline (PBS) and Ficoll-PaqueTM PLUS (1.077 g/mL, Amersham Biosciences, Freiburg, Germany) density gradient centrifugation (1578 × g, 30 minutes, 25°C). Harvested cells (1 × 10⁶ cells/mL) from the interface and plasma samples were stored at -80°C until RNA extraction.

Khalvati Fahlyani B., Behzad-Behbahani A., Taghavi S.A., Farhadi A., Salehi S., Adibzadeh S., Aboualizadeh F., Alavi P., Nikouyan N., Okhovat M.A., Ranjbaran R., Rafiei Dehbidi G.R, & Shakibzadeh A. (2015). Development of an In-House TaqMan Real Time RT-PCR Assay to Quantify Hepatitis C Virus RNA in Serum and Peripheral Blood Mononuclear Cells in Patients With Chronic Hepatitis C Virus Infection. Hepatitis Monthly, 15(8), e28895.

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Cytokine Profiling of Healthy PBMCs

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The research performed in this study followed the tenets of the Declaration of Helsinki and was approved by the Institutional Ethics Committee of Seoul National University Hospital. After written informed consent was obtained, PBMCs were obtained from 20 healthy individuals by density gradient centrifugation on Ficoll-Paque^TM PLUS (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The pellet was harvested and re-suspended for 5 min at 37° C in red blood cell lysis buffer (Sigma). To investigate cytokine production, PBMCs (2.5 × 10⁶ cells/ml) were treated with (or not) GV1001 (100 μM) and DHT (25 nM) for 24 h. Culture supernatant was assessed using Human Cytokine Array Q1 (RayBiotec, Norcross, GA, USA). Data were scanned by a gene microarray laser scanner and densitometry and analysis were performed using Q-Analyzer.

Kim Y., Lee D., Jo H., Go C., Yang J., Kang D, & Kang J.S. (2021). GV1001 interacts with androgen receptor to inhibit prostate cell proliferation in benign prostatic hyperplasia by regulating expression of molecules related to epithelial-mesenchymal transition. Aging (Albany NY), 13(3), 3202-3217.

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PBMC Isolation from Whole Blood

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Peripheral blood was collected from healthy unrelated donors into heparinized tubes. Blood was diluted (1:1) with PBS, layered onto 10 mL Ficoll-Paque^TM PLUS (Amersham Biosciences, Buckinghamshire, UK) into a 50 mL conical tube, and centrifuged (400× g; 30 min) without break. PBMNCs were collected, resuspended into cold PBS-BSA-EDTA, and centrifuged (300× g; 5 min). PBMNCs were plated at a high density into tissue-culture-treated culture dishes in fresh media (FM) for 30 min to allow full adherence of myeloid cells. Free-floating lymphocytes were finally collected, centrifuged, and counted. These cells were termed PBMC-enriched lymphocytes in the study.

Gauthier B.R., Rubio-Contreras D., Gómez-Rosado J.C., Capitán-Morales L.C., Hmadcha A., Soria B, & Lachaud C.C. (2022). Human Omental Mesothelial Cells Impart an Immunomodulatory Landscape Impeding B- and T-Cell Activation. International Journal of Molecular Sciences, 23(11), 5924.

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Prostate Cancer Biomarker Isolation

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Blood samples were collected at the Department of Urology in the First Affiliated Hospital of Zhejiang University from April 2014 to June 2015. When an abnormality is felt during a DRE, levels of PSA >4ng/mL, or abnormal findings in MRI, the patients who need prostate biopsy were included in this study. Patients considered with prostatitis, severe illness cannot withstand biopsy, were excluded. For each participant included in the study, 5 mL peripheral blood with anticoagulant was collected right when he was hospitalized. Then the blood samples were used for peripheral blood mononuclear cells (PBMCs) and serum isolation in 2 hours. PBMCs were isolated by Ficoll-Paque^TM Plus (Amersham Pharmacia Biotech, Shanghai, China) according to the facture's instructions. Collected PBMCs were stored at −80°C till used.

Mao Z., Ji A., Yang K., He W., Hu Y., Zhang Q., Zhang D, & Xie L. (2018). Diagnostic performance of PCA3 and hK2 in combination with serum PSA for prostate cancer. Medicine, 97(42), e12806.

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Culturing Diverse Epithelial Cell Lines

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Monkey (Cercopithecus aethiops) epithelial kidney cells (Vero E6, ATCC, CRL-1586), dog (Canis familiaris) epithelial kidney cells (MDCK, ATCC, CCL-34), and human lung epithelial cells (A549, ATCC CCL-185) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 1 g/L glucose (Gibco), supplemented with 10% heat-inactivated fetal bovine serum (HI‒FBS, Gibco) and 100 U/mL penicillin-streptomycin (PEST, Gibco) at 37°C in 95% air and 5% CO₂. Jurkat T-cells cells (ATCC, TIB-152) were maintained in RPMI 1640 medium (Fisher scientific, Austria) with 1.5 mM L-glutamine (Invitrogen, USA) and supplemented with 10% FBS. PBMC were isolated by the density gradient medium Ficoll-Paque^TM Plus (Amersham Biosciences, Sweden) according to the manufacturers’ instructions.

Omer A.A., Hinkula J., Tran P.T., Melik W., Zattarin E., Aili D., Selegård R., Bengtsson T, & Khalaf H. (2022). Plantaricin NC8 αβ rapidly and efficiently inhibits flaviviruses and SARS-CoV-2 by disrupting their envelopes. PLOS ONE, 17(11), e0278419.

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Isolation and Analysis of Heparinized PBMCs

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Heparinized PBMCs were isolated by Ficoll PaqueTM Plus (Amersham Pharmacia Biotech) density-gradient centrifugation, counted and stained with the appropriate combination of fluorescent labeled antibodies and analyzed by flow cytometry [22 (link)]. Dead cells were excluded from analysis by side/forward scatter gating. All analyses were performed on a LSRFortessa-X20 (BD Biosciences) interfaced to PC FACSDiva software. A total of 500,000 events per sample were analyzed.

Valentini D., Di Camillo C., Mirante N., Marcellini V., Carsetti R, & Villani A. (2020). Effects of Pidotimod on recurrent respiratory infections in children with Down syndrome: a retrospective Italian study. Italian Journal of Pediatrics, 46, 31.

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Isolation and Sorting of B Cell Subsets

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Peripheral blood mononuclear cells were isolated from heparinized peripheral blood by Ficoll-Paque^TM Plus (Amersham Biosciences, Little Chalfont, UK) density-gradient centrifugation and were stained with the following antibodies: clone ML5 (anti-CD24), clone M-T271 (anti-CD27), clone G18-145 (anti-IgG), and streptavidin-APC-Cy7 were obtained from BD Biosciences (San Diego, CA, USA) and clone B35C6B4 (anti-IgA₁) and clone A964D2 (anti-IgA₂) from Southern Biotechnologies. After staining the lymphocytes were sorted as mature-naive B cells (CD24⁺CD27⁻) and IgM memory B cells (CD24⁺CD27⁺IgG⁻IgA¹⁻²⁻) using a FACSvantage SE (Becton and Dickinson, Sunnyvale, California, USA). A negative gating strategy was used to sort IgM memory B cells in order to avoid B-cell activation through the BCR. Dead cells were excluded from analysis by side/forward scatter gating and cell purity was >98%. Cord blood mononuclear cells were stained for CD24 and CD38 and sorted for transitional B cells (CD24^brightCD38^bright) as described above.

Carsetti R., Di Sabatino A., Rosado M.M., Cascioli S., Piano Mortari E., Milito C., Grimsholm O., Aranburu A., Giorda E., Tinozzi F.P., Pulvirenti F., Donato G., Morini F., Bagolan P., Corazza G.R, & Quinti I. (2020). Lack of Gut Secretory Immunoglobulin A in Memory B-Cell Dysfunction-Associated Disorders: A Possible Gut-Spleen Axis. Frontiers in Immunology, 10, 2937.

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Isolation and Differentiation of Alternatively Activated Macrophages

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Human PBMCs (peripheral blood mononuclear cells) were isolated from buffy coats (NHS Blood and Transport) by centrifugation over a Ficoll‐Paque^TM PLUS (Amersham) gradient and monocytes isolated by adherence as previously reported. Autologous plasma was collected, heat‐inactivated (56℃, 30 min) and used to supplement (1%) X‐VIVO10 (Lonza) medium to produce complete growth medium. Cells were differentiated for 3 days (37℃, 5% CO₂) in complete growth medium +25 ng/ml recombinant human IL‐4 (rhIL‐4, Peprotech) to generate alternatively activated macrophages [14 , 21 (link)]. The use of human blood was approved by the NHS National Research Ethics Service (09/H0606/3).

Sayce A.C., Martinez F.O., Tyrrell B.E., Perera N., Hill M.L., Dwek R.A., Miller J.L, & Zitzmann N. (2021). Pathogen‐induced inflammation is attenuated by the iminosugar MON‐DNJ via modulation of the unfolded protein response. Immunology, 164(3), 587-601.

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Myeloma Patient Sample Collection and Processing

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BM aspirates and PB samples were obtained from patients with MGUS [BM: N = 5, PB: N = 5] and SMM [BM: N = 5, PB: N = 5] and patients with MM (newly diagnosed [BM: N = 18, PB: N = 10], relapsed [BM: N = 14, PB: N = 12], relapsed/refractory [BM: N = 18, PB: N = 12]) after informed consent, in accordance with the Declaration of Helsinki, and with approval by the Institutional Review Board at Dana-Farber Cancer Institute (Boston, MA). In addition, healthy individuals BM [N = 5] or leukapheresis [N = 12] products were purchased from either AllCells (Alameda, CA) or the Blood Donor Center at Boston Children’s Hospital (Boston, MA), respectively. Mononuclear cells were isolated from BM (BMMC) or PB (PBMC) by standard density gradient centrifugation using Ficoll-Paque^TM Plus (Amersham Pharmacia Biotech AB, Uppsala Sweden) and used in these studies.

Bae J., Accardi F., Hideshima T., Tai Y.T., Prabhala R., Shambley A., Wen K., Rowell S., Richardson P.G., Munshi N.C, & Anderson K.C. (2021). Targeting LAG3/GAL-3 to overcome immunosuppression and enhance anti-tumor immune responses in multiple myeloma. Leukemia, 36(1), 138-154.

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