HEK 293T cells were obtained from ATCC (CRL-3216). PBMCs were isolated by Ficoll-Hypaque density centrifugation (Amersham Pharmacia Biotech) from cytopheresis or whole-blood samples obtained from healthy volunteers and patients, respectively. EBV-immortalized LCLs (EBV-B cells) were obtained as previously described (Dupuis et al., 2001 (link)). Primary keratinocyte isolation: Punch biopsy specimens from the inner upper arm were washed three times with serum-free DMEM supplemented with 50 µg/ml gentamycin. They were then treated with dispase (354235; BD) at 4°C overnight. The following day, dermis and epidermis were separated manually and treated with Accutase (A6964; Sigma-Aldrich). The epidermis was then cut into pieces and wounded to obtain basal keratinocytes, which were cultured as described below.
Ficoll hypaque density centrifugation
Manufactured by Cytiva
Sourced in Sweden
Ficoll-Hypaque density centrifugation is a laboratory technique used for the separation and isolation of specific cell types from a complex mixture, such as blood or tissue samples. It utilizes a density gradient medium to facilitate the separation of cells based on their density differences during centrifugation.
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Lab products found in correlation
Fixation permeabilization kit, by Thermo Fisher Scientific (2 mentions)
Rhil 3, by Miltenyi Biotec (2 mentions)
Aqua dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Phospho flow pe mouse anti p stat5 py694 antibody, by BD (2 mentions)
96 well 5 bottom plates, by Thermo Fisher Scientific (2 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (2 mentions)
Gallios flow cytometer, by Beckman Coulter (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dispase, by BD (1 mentions)
Accutase, by Merck Group (1 mentions)
Gm csf, by Miltenyi Biotec (1 mentions)
Rhgm csf, by Miltenyi Biotec (1 mentions)
Cd4 fitc, by Sony (1 mentions)
5 protocols using ficoll hypaque density centrifugation
1
Primary Cell Culture Protocols
2
HTLV-1 Infection and HAM/TSP Diagnosis
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Ten HAM/TSP patients who tested positive for the virus were recruited from the HTLV-1 outpatient clinic of the Emilio Ribas Institute for Infectious Diseases. At the time of blood donation, the eight individuals of the ASP group were found to be HTLV-1 carriers. Five HCs were selected from a group of apparently healthy volunteers recruited by laboratory personnel who did not have HTLV-1 infection. Thus, the total sample included ten HAM/TSP cases, eight ASP cases, and five HCs. The enzyme immunoassays Murex HTLV I + II (Abbott/Murex, Wiesbaden, Germany) and Vironostika HTLV-I/II (BioMérieux bv, Boxtel, The Netherlands) were used to diagnose HTLV-1 infection and HTLV BLOT 2.4 (Genelabs Diagnostics, Science Park, Singapore) to confirm infection. The world health organization (WHO) criteria for HTLV-1-associated disease were used to determine the clinical status of HAM/TSP patients [24 ]. Whole blood samples were collected and processed after approval by the Ethics Committee of the Institute of Health (CAPPesq, approval number 56/2012). All subjects who participated in the study also signed a written informed consent form. PBMCs were isolated by Ficoll-Hypaque density centrifugation (Amersham, Upsala, Sweden) and subsequently stored as previously described [25 (link)].
3
PBMC Isolation and Phospho-STAT5 Analysis
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Human peripheral blood mononuclear cells (PBMCs) from a healthy donor were isolated from whole blood by Ficoll- Hypaque density centrifugation (Amersham-Pharmacia- Biotech). The cells were counted and plated at 2 × 106 cells/well in 96-well V-bottom plates (Thermo-Fisher-Scientific), in 100 μL of RPMI (GibcoBRL, Invitrogen) supplemented with 10% fetal bovine serum (GibcoBRL, Invitrogen), or 100 μL of RPMI supplemented with 1:10 serafrom patients or controls. PBMCs were left unstimulated or were stimulated with 10 ng/μL of rhIL-3 or GM-CSF or 50 ng/μL of rhIL-3 (Miltenyi-Biotec) for 15 min at 37°C. Thereafter, cells were fixed and permeabilized with a fixation/permeabilization kit (eBioscience). Extracellular labeling was performed with antibodies anti CD14-Pacific Blue and anti CD4-FITC (Sony-Biotechnology, clones M5E2 and RPA-T4, respectively). Cell viability was determined with the Aqua Dead Cell Stain Kit (Thermo-Fisher-Scientific). STAT5 phosphorylation (p-STAT5) levels were assessed by intracellular staining with Phospho-Flow PE Mouse Anti-p-STAT5 (pY694) antibody (BD Biosciences). Data were collected with a Gallios flow cytometer (Beckman-Coulter) and analyzed with FlowJo software v.10.6.2 (Becton–Dickinson).
4
ADCC Assay with HIV-Negative PBMCs
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Blood samples were collected and centrifuged to separate plasma. For ADCC assays, plasma was first diluted (10-fold in RPMI medium), passed through a 0.2-μm-pore-size filter, and heat inactivated (1 h at 56°C). PBMCs from one HIV-negative donor were isolated by Ficoll-Hypaque density centrifugation (Amersham, Sweden), cryopreserved, and used as effector cells in ADCC assays. Cells from the same donor were used in all assays to avoid bias from donor to donor.
5
Quantifying STAT5 Phosphorylation in PBMC Subsets
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Human peripheral blood mononuclear cells (PBMCs) from healthy controls were isolated from whole blood by Ficoll-Hypaque density centrifugation (Amersham-Pharmacia-Biotech, Sweden). The cells were counted and plated at 2 × 106 cells/well in 96-well V-bottom plates (Thermo-Fisher-Scientific), in 100 µL RPMI (GibcoBRL, Invitrogen), supplemented with 10% fetal bovine serum (FBS) (GibcoBRL, Invitrogen) or 100 µL RPMI supplemented with 10% plasma from patients or controls. PBMCs were left unstimulated or were stimulated with 5 to 80 ng/mL rhGM-CSF or 100 ng/mL rhIL-3 (Miltenyi-Biotec) for 30 min at 37°C, and the cells were then fixed permeabilized with a fixation/permeabilization kit (eBioscience). Extracellular labeling was performed with CD14-Pacific Blue and CD4-FITC (Sony-Biotechnology, clones M5E2 and RPA-T4, respectively). Cell viability was determined with the Aqua Dead Cell Stain Kit (Thermo-Fisher-Scientific), and STAT5 phosphorylation (p-STAT5 levels) was assessed by intracellular staining with Phospho-Flow PE Mouse Anti-p-STAT5 (pY694) antibody (BD-Biosciences). Data were collected with a Gallios flow cytometer (Beckman-Coulter) and analyzed with FlowJo software v.10.6.2 (Becton–Dickinson).
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