Ficoll paque density gradient centrifugation

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Ficoll-Paque density gradient centrifugation is a laboratory technique used for the separation and isolation of cells, organelles, and other biological particles based on their density differences. It utilizes a specialized liquid medium called Ficoll-Paque, which forms a density gradient during centrifugation. This allows the selective separation of specific cell types or components from a heterogeneous sample.

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Lab products found in correlation

Recovery cell culture freezing medium, by Thermo Fisher Scientific (1 mentions) Cryostor cs10, by Merck Group (1 mentions) Glutamine, by Thermo Fisher Scientific (1 mentions) Dnase 1, by Roche (1 mentions) Liberase tl, by Roche (1 mentions) Cobas ampliprep cobas taqman v2, by Roche (1 mentions) Dynabeads untouched human monocytes kit, by Thermo Fisher Scientific (1 mentions) Bca method, by Merck Group (1 mentions) Protease inhibitor cocktail tablet, by Roche (1 mentions) Prism 9, by GraphPad (1 mentions)

5 protocols using ficoll paque density gradient centrifugation

Ebola Survivor and Control Samples Protocol

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We studied samples from patients in whom EVD was diagnosed during the 2013–2016 outbreak. We collected serum samples from 77 uninfected controls and 101 EVD survivors in Guéckédou, Guinea, during May 2015–September 2017. In addition, the European Mobile Laboratory provided DNA isolated from whole blood samples of 119 persons who had died of EVD.
We defined EVD survivors as EBOV-infected patients who had survived the acute phase of EVD and were discharged from the Ebola treatment center in Guéckédou after testing negative for Ebola 4 times by reverse transcription PCR. The survivors in our study included only persons with an original certificate of survivorship issued by the Guinean government. Controls tested negative for EBOV-specific antibodies and EBOV neutralizing antibodies in plasma.
We collected blood samples in EDTA tubes for routine blood tests, serologic assays for EBOV antigen, and nucleic acid detection of EBOV RNA by reverse transcription PCR. Afterward, we isolated peripheral blood mononuclear cells (PBMCs) from whole blood samples using Ficoll-Paque density gradient centrifugation (Sigma-Aldrich Inc., https://www.sigmaaldrich.com) according to the manufacturer’s instructions. To store PBMCs at room temperature, we used DNAgard Blood (Sigma-Aldrich Inc.) according to the manufacturer’s instructions.

Wawina-Bokalanga T., Vanmechelen B., Lhermitte V., Martí-Carreras J., Vergote V., Koundouno F.R., Akoi-Boré J., Thom R., Tipton T., Steeds K., Moussa K.B., Amento A., Laenen L., Duraffour S., Gabriel M., Ruibal P., Hall Y., Kader-Kondé M., Günther S., Baele G., Muñoz-Fontela C., Van Weyenbergh J., Carroll M.W, & Maes P. (2021). Human Diversity of Killer Cell Immunoglobulin-Like Receptors and Human Leukocyte Antigen Class I Alleles and Ebola Virus Disease Outcomes. Emerging Infectious Diseases, 27(1), 76-84.

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Profiling CD8+ T Cells from Synovial Tissue

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Whole blood was collected and PBMCs were isolated using Ficoll-Paque density gradient centrifugation (Sigma Aldrich, GE17-1440-03) for cellular profiling of CD8⁺ T cells. Cells were cryopreserved in Recovery Cell Culture Freezing Medium (Thermo Fisher Scientific, 12648010). For synovial tissue disaggregation, cryopreserved synovial tissues in Cryostor CS10 (Sigma-Aldrich, C2874-100ML) were obtained from hospital for special surgery. Each synovial tissue specimen was fully thawed at 37 °C and rinsed with RPMI 1640 (Corning Life Science, MT15040CV) containing 10% FBS (ATCC, 30-2020) and 1% glutamine (Gibco, 25030081). The tissue fragments were minced and digested with 100 μg/mL Liberase TL (Roche, 05401020001) and 100 μg/mL DNase I (Roche, 4716728001) in RPMI 1640 for 30 min at 37 °C with inverting to disaggregate the fragments into single cell suspensions. The digested fragments were filtered through a 70 μm cell strainer and washed with cold RPMI 1640 with 10% FBS and 1% glutamine before antibody staining and 10X single cell RNA sequencing.

Moon J.S., Younis S., Ramadoss N.S., Iyer R., Sheth K., Sharpe O., Rao N.L., Becart S., Carman J.A., James E.A., Buckner J.H., Deane K.D., Holers V.M., Goodman S.M., Donlin L.T., Davis M.M, & Robinson W.H. (2023). Cytotoxic CD8+ T cells target citrullinated antigens in rheumatoid arthritis. Nature Communications, 14, 319.

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HIV Reservoir Characterization in Viremic and Suppressed Donors

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The study was approved by the University of Pittsburgh Institutional
Review Board. Study participants were recruited from the University of
Pittsburgh AIDS Center for Treatment and provided written informed consent. Two
groups of participants were enrolled: (1) viremic donors with plasma HIV-1 RNA
>1,000 copies/ml (Roche COBAS AmpliPrep/COBAS TaqMan, v2.0) who were not
currently receiving ART (some participants had a history of ART exposure); and
(2) virologically suppressed donors who had been on stable suppressive ART for
at least 6 months with plasma HIV-1 RNA <20 copies/ml (Roche). In a
subset of donors, samples were collected at two timepoints (5 of 12 viremic
donors and 19 of 23 virologically suppressed donors).
Sample specimens were collected through either large-volume phlebotomy
(100 –180 ml) or leukapheresis, and then processed to peripheral blood
mononuclear cells (PBMC) by Ficoll-Paque density gradient centrifugation
(Sigma-Aldrich, USA) within 4 hours of collection. The cells were cryopreserved
in 5 –10 million aliquots and stored in liquid nitrogen before analysis.
Plasma was harvested from whole blood by double centrifugation (400 g ×
10 min, followed by 1,350 g × 15 min) and stored at
−80°C before analysis.

Hong F., Jacobs J.L., Aga E., Cillo A.R., Fyne E., Koontz D.L., Zheng L, & Mellors J.W. (2018). Associations between HIV-1 DNA copy number, proviral transcriptional activity, and plasma viremia in individuals off or on suppressive antiretroviral therapy. Virology, 521, 51-57.

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Isolation and Characterization of Human Monocytes

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Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-Paque density gradient centrifugation (Sigma-Aldrich), and then washed three times with phosphate buffer saline (PBS). Monocytes were purified by negative selection using Dynabeads Untouched Human Monocytes Kit (Invitrogen). After that, cells were washed with PBS and treated with lysis solution for LC-MSE supplemented with protease inhibitor cocktail tablets (Roche). Extracts were sonicated for 5 minutes, centrifuged at 15,000 × g for 15 minutes at 4 °C, and the supernatants were recovered. Protein concentration for each extraction was determined by BCA method (Sigma-Aldrich), and samples were stored at −80 °C.

Echevarria-Lima J., de Abreu Pereira D., de Oliveira T.S., de Melo Espíndola O., Lima M.A., Celestino Leite A.C., Sandim V., Rodrigues Nascimento C., E. Kalume D, & B. Zingali R. (2018). Protein Profile of Blood Monocytes is Altered in HTLV-1 Infected Patients: Implications for HAM/TSP Disease. Scientific Reports, 8, 14354.

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Bone Marrow Mononuclear Cell Isolation and Analysis

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Aspiration of fresh bone marrow was approved by the Institutional Review Board (PV5505). Fresh bone marrow aspirates were collected from nine newly diagnosed, untreated multiple myeloma patients. Ficoll-Paque density gradient centrifugation (Sigma-Aldrich, St. Louis, MO, USA) was carried out to isolate bone marrow mononuclear cells (BM-MNCs). Remaining erythrocytes were depleted by resuspending the resulting cell pellet in red cell lysis buffer (NH4Cl + KHCO3 + EDTA).
BM-MNCs were stained using PacO and a panel of fluorochrome-labeled antibodies targeting CD19, CD38, CD39, CD45, CD55, CD56, CD59, CD138, CD229, CD319 and analyzed by flow cytometry to determine the degree of bone marrow infiltration with malignant plasma cells (27 (link)). Multiple myeloma cells were identified by high expression of CD38.
Blocking assays were carried out by incubating BM-MNCs without (positive control) or with 100 nM of daratumumab or JK36-hcAb in PBS/BSA at 4 °C for 30 min. After one washing step, detection nanobody JK36^AF680 was added for another 30 min. Mean fluorescence intensities (MFI) of bound nanobody JK36^AF680 were assessed by flow cytometry. Relative fluorescence intensities (%) of multiple myeloma cells labeled with nanobody JK36^AF680 were calculated as follows:
Significant differences in relative fluorescence intensities were calculated by using one-way ANOVA (GraphPad Prism 9.3.1).

Pape L.J., Hambach J., Gebhardt A.J., Rissiek B., Stähler T., Tode N., Khan C., Weisel K., Adam G., Koch-Nolte F, & Bannas P. (2022). CD38-specific nanobodies allow in vivo imaging of multiple myeloma under daratumumab therapy. Frontiers in Immunology, 13, 1010270.

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