Blood mononuclear cells (BMCs) were separated on Ficoll-Histopaque density gradients (Sigma-Aldrich Co., St Louis, MO, USA) according to the manufacturer’s protocol,9 (link) with minor modifications. PBMCs were separated from 3 mL of whole blood using centrifugation on a Ficoll–Histopaque gradient at 2200 rpm for 30 minutes. The PBMC layer was separated and washed 3 times with 1× phosphate buffered saline with a pH of 7.4 at 2200 rpm for 10 minutes. The cells were resuspended in fresh RPMI 1640 (Cellgro; CellGenix GmbH, Feiburg, Germany), and 10 μL of the cell suspension was mixed with an equal volume of trypan blue dye. This mixture was used for cell counting and viability determination using an automatic Countess machine (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA). The cells were preserved with 10% fetal bovine serum, 1% each of penicillin, streptomycin, and gentamycin, and 1% nonessential amino acids (Sigma-Aldrich Co.), and they were stored at −80°C until further use.
Ficoll histopaque density gradient
Manufactured by Merck Group
Sourced in United States
Ficoll Histopaque density gradient is a laboratory product used for the separation and isolation of biological cells, such as mononuclear cells, from whole blood or other complex samples. It utilizes a density gradient medium to facilitate the separation of different cell types based on their density differences.
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Lab products found in correlation
Urea, by Bio-Rad (2 mentions)
Iodoacetic acid, by Merck Group (2 mentions)
Thiourea, by Bio-Rad (2 mentions)
Mineral oil, by Bio-Rad (2 mentions)
Sodium dodecyl sulfate (sds), by Bio-Rad (2 mentions)
Bis acrylamide, by Merck Group (2 mentions)
Pierce 660 nm protein assay kit, by Thermo Fisher Scientific (2 mentions)
Proteomics grade trypsin from porcine pancreas, by Merck Group (2 mentions)
Chaps 3 3 cholamidopropyl dimethylammonio 1 propanesulfonate, by Bio-Rad (2 mentions)
Anti age monoclonal antibodies for western blotting, by MP Biomedicals (2 mentions)
C 18 ziptip columns, by Merck Group (2 mentions)
Dithiothreitol (dtt), by Bio-Rad (2 mentions)
Ipg strip, by Bio-Rad (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (2 mentions)
Ammonium bicarbonate, by Merck Group (2 mentions)
Acrylamide, by Merck Group (2 mentions)
Gentamycin, by Merck Group (1 mentions)
Non essential amino acids (neaa), by Merck Group (1 mentions)
Rpmi 1640, by CellGenix (1 mentions)
9 protocols using ficoll histopaque density gradient
1
Cryopreservation of Peripheral Blood Mononuclear Cells
2
Isolating PBMCs from Whole Blood
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PBMCs were separated on Ficoll-Histopaque density gradients (Sigma-Aldrich Co., St Louis, MO, USA) according to the manufacturer’s protocol, (Thimme R, Bukh J, Spangenberg HC, et al. Viral and immunological determinants of hepatitis C virus clearance, persistence, and disease. Proc Natl Acad Sci U S A. 2002;99(24 (link)):15661–15668.) with minor modifications. PBMCs were separated from 3 mL of fresh EDTA venous blood using centrifugation on a Ficoll–Histopaque gradient at 2200 rpm for 30 minutes. The PBMC layer was separated and washed three times with 1× phosphate buffered saline (PBS) with a pH of 7.4 at 2200 rpm for 10 minutes. The cells were resuspended in 1x PBS, and they were stored at −80 ºC until used.
3
Lymphocyte Isolation and Caspase-3 Assay
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Lymphocytes were isolated from each blood sample using Ficoll-Histopaque density gradients (Sigma, St. Louis, MO, USA) with modification. Blood was diluted 1 : 3 with phosphate buffered saline (PBS) and layered on to the Histopaque in the ratio of 2 : 1 (blood + PBS: Histopaque). The blood was centrifuged at 400 ×g for 20 min at room temperature. The lymphocytes layer was removed and then washed twice in PBS at 250 ×g for 10 min each. Liquid layer was removed and then 1 mL of PBS was added to sediment layer (lymphocytes layer) as a final sample. A thin layer of final sample was prepared on a glass slide and number of lymphocytes was counted by microscope (Olympus Optical Co. Ltd., Japan). LC was also expressed as 106 cells/mL.
Lymphocytes collected in previous step were used for caspase-3 activity assay according to manufacturer's instructions. The Caspase-3 Colorimetric Assay Kit provides a means for assaying the activity of caspases that recognize the sequence DEVD. The assay is based on spectrophotometric detection of the chromophore p-nitroaniline (pNA) after cleavage from the labeled substrate DEVD-pNA. The pNA light emission can be quantified using a microtiter plate reader at 405 nm. The caspase-3 activity is presented as pNA optical density (OD)/106 cells per mL.
Lymphocytes collected in previous step were used for caspase-3 activity assay according to manufacturer's instructions. The Caspase-3 Colorimetric Assay Kit provides a means for assaying the activity of caspases that recognize the sequence DEVD. The assay is based on spectrophotometric detection of the chromophore p-nitroaniline (pNA) after cleavage from the labeled substrate DEVD-pNA. The pNA light emission can be quantified using a microtiter plate reader at 405 nm. The caspase-3 activity is presented as pNA optical density (OD)/106 cells per mL.
4
Isolation of Human Neutrophils from Blood
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Human neutrophils from healthy blood were obtained by centrifugation on Ficoll Histopaque density gradient (Sigma-Aldrich), followed by hypotonic lysis (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.4) to remove red blood cells. Isolated neutrophils (≥95% of the cells) were washed in PBS and resuspended in RPMI. All the procedures dealing with human blood were performed in accordance with the guidelines of the Research Ethics Committee (Hospital Universitário Fraga Filho, UFRJ, Brazil), protocol number: 4261 015400005257.
5
Comprehensive Proteomics Sample Preparation
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All reagents used were of molecular grade, protease inhibitor cocktail, Phenylmethylsulfonyl fluoride (PMSF), Proteomics grade trypsin from porcine pancreas, Iodoacetic acid (IAA), Acrylamide, bis Acrylamide, ficoll histopaque density gradient and Ammonium bicarbonate were procured from Sigma-Aldrich, St. Louis, MO, USA. Sodium Dodecyl Sulfate (SDS), IPG strips, Urea, thioUrea, Mineral oil, Dithiothreitol (DTT), CHAPS (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate) were purchased from Bio-Rad Laboratories, Inc. California, USA. For protein quantification, Pierce™ 660nm Protein Assay kit from Thermo Fisher Scientific Inc. USA was used. Mouse, Anti-AGE monoclonal antibodies for western blotting were obtained from MP Biomedicals, USA. The polyvinylidenedifluoride (PVDF) membrane (0.45μm) and dialysis tubing were purchased from Millipore Corporation, MA, USA. C-18 ZipTip columns for concentrating and desalting the peptides were purchased from Millipore, Billerica, MA, USA. Pre-stained protein ladder was procured from Real Biotech Corporation, Taipei, Taiwan.
6
Immune Cell Profiles Across Age and CMV
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We studied 32 healthy donors; 20 young individuals (18–35 years old) stratified by CMV serostatus –10 CMV-seronegative (4 male/6 female; mean age 26.3, SD = 5.8) and 10 CMV-seropositive (6 male/4 female; mean age 27, SD = 4.3) – and 12 middle age individuals all of them CMV-seropositive (3 male/9 female; 40–60 years old, mean age 50.25, SD = 4.3). Due to the high prevalence of CMV in Spain we could not recruit enough middle age CMV-seronegative individuals (over 80% in older than 40 years old [2] (link)). All subjects studied met the following exclusion criteria: absence of diabetes, cancer, severe renal failure, severe liver disease, endocrine disorders, autoimmune diseases, or acute infectious disease; they were not consuming drugs whose activity is known to modify the functions of the immune system.
Peripheral blood from each subject was collected by venipuncture in Lithium Heparin-containing tubes. Within 1–3 h after collection PBMCs were isolated from each blood sample using Ficoll-Histopaque density gradient centrifugation (Sigma Aldrich. Steinheim, Germany) and cryopreserved until the time the experiments were performed.
Peripheral blood from each subject was collected by venipuncture in Lithium Heparin-containing tubes. Within 1–3 h after collection PBMCs were isolated from each blood sample using Ficoll-Histopaque density gradient centrifugation (Sigma Aldrich. Steinheim, Germany) and cryopreserved until the time the experiments were performed.
7
Monocyte-Derived Human Dendritic Cells
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Immature monocyte-derived hDCs were obtained from freshly isolated healthy peripheral blood monocytes using Ficoll–Histopaque density gradient separation (Sigma-Aldrich, Saint Louis, MO, USA). Peripheral blood mononuclear cells (PBMCs) were washed three times, and the CD14+ cell population was enriched by positive selection using magnetic cell sorting (Miltenyi Biotec, Leiden, The Netherlands) to isolate the CD14+ subset. Cells were then resuspended and plated in RPMI medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF) (50 ng/mL) and interleukin 4 (IL4) (100 UI/mL) (PeproTech, Rocky Hill, NJ, USA) for 7 days.
8
Evaluating SARS-CoV-2 Infection in A549 Cells
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Peripheral blood mononuclear cells (PBMC) were isolated by a Ficoll-histopaque density gradient method (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) from each healthy donor (n = 3-7). The A549 cell line was grown in Dulbecco’s Modified Eagle Medium (DMEM, Sigma-Aldrich, St Louis, MO, USA) supplemented with 2% heat-inactivated fetal bovine serum (FBS) (GIBCO- Thermo Fisher Scientific Inc, Waltman, MA, USA), 2mM L-glutamine (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) and 1% penicillin-streptomycin at 37°C with 5% CO2. The viral stock was produced from a Colombian SARS-CoV-2 isolated: D614G strain (EPI_ISL_536399) (40 (link)). The virus was used at 0.1 multiplicity of infection (MOI) and incubated for 8, 24 or 48h at 37°C with 5% CO2 (according to the experimental results). Unexposed cells were used as a negative control.
9
Proteomic Analysis of Advanced Glycation End-products
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All reagents used were of molecular grade, protease inhibitor cocktail, Phenylmethylsulfonyl fluoride (PMSF), Proteomics grade trypsin from porcine pancreas, Iodoacetic acid (IAA), Acrylamide, bis Acrylamide, ficollhistopaque density gradient and Ammonium bicarbonate were procured from Sigma-Aldrich, St. Louis, MO, USA. Sodium Dodecyl Sulfate (SDS), IPG strips, Urea, thioUrea, Mineral oil, Dithiothreitol (DTT), CHAPS (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate) were purchased from Bio-Rad Laboratories, Inc. California, USA. For protein quantification, Pierce™ 660nm Protein Assay kit from Thermo Fisher Scientific Inc. USA was used. Mouse, Anti-AGE monoclonal antibodies for western blotting were obtained from MP Biomedicals, USA. The polyvinylidenedifluoride (PVDF) membrane (0.45µm) and dialysis tubing were purchased from Millipore Corporation, MA, USA. C-18 ZipTip columns for concentrating and desalting the peptides were purchased from Millipore, Billerica, MA, USA. Pre-stained protein ladder was procured from Real Biotech Corporation, Taipei, Taiwan. All other reagents used were of high analytical grade.
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