PBLs were isolated using a Ficoll-1077 (C-44010, Sigma) density gradient. Isolated PBLs were washed three times with PBS, suspended in RPMI 1640 (containing 10% FBS, 2 mM L-glutamine, and 1% penicillin/streptomycin), and cultivated at 37°C in a humidified atmosphere comprising 5% CO2. The HeLa cells were cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin at 37°C in a humidified atmosphere comprising 5% CO2.
Ficoll 1077
Manufactured by Merck Group
Sourced in United States
Ficoll 1077 is a high-molecular-weight, sucrose-epichlorohydrin copolymer used in density gradient centrifugation for the isolation and purification of cells, cellular organelles, and other biological particles. It has a density of approximately 1.077 g/mL.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi medium, by Sartorius (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Ecl solution, by Cytiva (1 mentions)
Enhancer solution, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Sartorius (1 mentions)
Fetal calf serum (fcs), by Sartorius (1 mentions)
Methylated bovine serum albumin (mbsa), by Merck Group (1 mentions)
Complete freund s adjuvant cfa, by Merck Group (1 mentions)
Protein a sepharose beads, by Cytiva (1 mentions)
Ecori, by New England Biolabs (1 mentions)
Facscanto, by BD (1 mentions)
Trypan blue test, by Thermo Fisher Scientific (1 mentions)
Pbs 1x, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharide lps, by InvivoGen (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Ficoll, by BD (1 mentions)
8 protocols using ficoll 1077
1
Isolation and Culture of Peripheral Blood Lymphocytes
2
Comprehensive Materials Acquisition Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Amplification buffer was obtained from Invitrogen (CA, USA); Dextran T-500 was obtained from Pharmacosmos A/S (Denmark); Dulbecco’s Modified Eagle’s Medium (DMEM) was obtained from Biological Industries (Beit Haemek, Israel); ECL solution was obtained from Amersham Biosciences (Buckinghamshire, UK); EcoRI was obtained from New England Biolabs; enhancer solution was obtained from Invitrogen; EX-CELL® 293 medium was obtained from SAFC Biosciences; fetal calf serum (FCS) was obtained from Biological Industries (Beit Haemek, Israel); Ficoll 1077 was obtained from Sigma-Aldrich (Israel).
Freund’s complete adjuvant (CFA) was obtained from Sigma-Aldrich or Difco; FuGENE® 6 was obtained from Roche; methylated bovine serum albumin (mBSA) was obtained from Sigma-Aldrich; NotI was obtained from New England Biolabs; NuPAGE® Bis-Tris gels were obtained from Invitrogen; the pIRESpuro3 vector was obtained from BD Biosciences Clontech; Protein A-Sepharose® beads were obtained from Amersham; RPMI medium was obtained from Biological Industries (Beit Haemek, Israel); Taq polymerase (Platinum® Pfx DNA polymerase) was obtained from Invitrogen; VCAM-1 (human) was obtained from R&D Systems, Inc. (Minneapolis, MN, USA); and all the recombinant human chemokines were ordered from PeproTech, Inc. (Rocky Hill, NJ, USA).
Freund’s complete adjuvant (CFA) was obtained from Sigma-Aldrich or Difco; FuGENE® 6 was obtained from Roche; methylated bovine serum albumin (mBSA) was obtained from Sigma-Aldrich; NotI was obtained from New England Biolabs; NuPAGE® Bis-Tris gels were obtained from Invitrogen; the pIRESpuro3 vector was obtained from BD Biosciences Clontech; Protein A-Sepharose® beads were obtained from Amersham; RPMI medium was obtained from Biological Industries (Beit Haemek, Israel); Taq polymerase (Platinum® Pfx DNA polymerase) was obtained from Invitrogen; VCAM-1 (human) was obtained from R&D Systems, Inc. (Minneapolis, MN, USA); and all the recombinant human chemokines were ordered from PeproTech, Inc. (Rocky Hill, NJ, USA).
3
CX3CR1 Expression in PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CX3CR1 expression levels by peripheral blood mononuclear cells (PBMC) were examined. Heparinized blood samples were collected and placed on ice. The samples were then processed with Ficoll 1077 (sigma, USA) and washed with phosphate buffered saline for twice. Antibody staining with anti human CX3CR1 PE (2A9-1), anti human CD4 FITC (RPA-T4), anti human CD8a PerCP (SK 1), anti human CD16 APC (CB16) from eBioscience (San Diego, California, USA) (San Diego, CA) was performed at 4°C with a predetermined optimal concentration of the test monoclonal Ab for 20 min. After staining with antibody, the product was aliquoted to 1×106; 5×105 cells per tubes. Cells were washed and analyzed with a Fluorescence-activated cell sorting (FACS) scan flow cytometer. FACS analysis was performed on a FACS Canto (BD Biosciences, San Diego, CA). Positive and negative populations of cells were determined with unreactive isotype matched monoclonal Abs (Beckman Coulter Korea Ltd., Seoul, Korea) as controls for background staining.
4
Isolation and Viability Assessment of Lymphocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To perform the comet assay the lymphocytes were isolated by Ficoll-1077 (Sigma Aldrich, St. Louis, MO, USA) density gradient centrifugation and washed in phosphate buffered saline (PBS 1X) (Gibco, Life Technologies, Nebraska, USA). The viability of the cells was tested by trypan blue test (Life Technologies, Nebraska, USA) and was kept greater than 90 %. The volume of cell suspension used in the test was 4×103 lymphocytes.
5
Isolation of Bone Marrow Mononuclear Cells
6
PBMC Isolation and Stimulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMCs were isolated by density centrifugation gradient (Ficoll 1077, Sigma-Aldrich, St. Louis, MO, USA) and cryopreserved in RPMI-1640 with fetal bovine serum (20%), dimethyl sulphoxide (10%), and penicillin/streptomycin (1%), in liquid nitrogen. PBMCs (3–5) × 106) were thawed in complete media (RPMI-1640 with 10% FBS, 2% HEPES, 2% l -Glutamine, 1% Pen/Strep) and incubated in triplicate wells with phosphate buffered saline (PBS; 100 μL/well) (Sigma-Aldrich, St. Louis, MO, USA) as negative control, lipopolysaccharide (LPS; 10 ng/mL) (Invivogen, San Diego, CA, USA) as positive control, or recombinant interferon-γ (IFN-γ; 150 U/mL) (Genentech, Inc., San Francisco, CA, USA).
7
PBMC Extraction from SARS-CoV-2 Patient Blood
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The PBMC samples (Ficoll preparation) were extracted from the fresh blood of the twenty SARS-CoV-2 patients. Peripheral blood samples (4 ml) from each patient were drawn into vacutainer tubes by using Dipotassium (K2) Ethylene Diamine Tetraacetic Acid (EDTA) as an anti-coagulant within the vacutainers. The Ficoll 1.077 (Sigma Aldrich) density gradient centrifugation method separated the PBMC samples. The blood was diluted with 1 × phosphate-buffered saline (PBS) 1:1 and was shifted to the Ficoll tube. After centrifugation (20 min, 1,000 × g at room temperature), the buffy coat of PBMCs was pooled and moved into a 15-ml falcon tube. The cells were then washed twice with 10 ml PBS and centrifuged at 250 × g for 10 min.
8
Isolation of Peripheral Blood Mononuclear Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After obtaining the consent of the subject's parents, 5ml of peripheral blood was collected from the patient in a relaxed state (no asthma attack). It was poured into a test tube containing EDTA anticoagulant (50mM) and was transferred to the laboratory. Then a concentration gradient of Ficoll 1.077 (Sigma-Aldrich, USA) was used to separate peripheral blood mononuclear cells (PBMC). 2.5 ml Ficoll was added to an 18 ml Falcon tube, and whole blood was gently added via Pipette Pasteur. Then centrifuge at 400°C for 20 minutes at room temperature to gently remove PBMC. The cells were washed three times with PBS and centrifuged at 250°C for 10 minutes. Then, to fit the number of cell populations in each sample, the separated PBMC was added to 1 ml with RPMI1640, and the number of cells was counted using a Neobar slide and adjusted to 4 million cells.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!