Peripheral blood samples were collected from the vena orbitalis, heparinized, and purified by centrifugation on a Ficoll-Hypaque Premium (GE Healthcare, Pittsburgh, PA, USA).
Ficoll hypaque premium
Manufactured by GE Healthcare
Sourced in United States
Ficoll-Hypaque PREMIUM is a density gradient medium used for the separation and purification of cells, such as mononuclear cells, from whole blood or other biological samples. It is a sterile, non-pyrogenic solution that can be used in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Dextran t500, by Merck Group (1 mentions)
Facsverse flow cytometer, by BD (1 mentions)
L ascorbic acid 2 phosphate, by Thermo Fisher Scientific (1 mentions)
Dexamethasone (dex), by Thermo Fisher Scientific (1 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (1 mentions)
Igf 1, by Thermo Fisher Scientific (1 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (1 mentions)
Insulin, by Thermo Fisher Scientific (1 mentions)
Transferrin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Harvard Bioscience (1 mentions)
Penicillin, by Sangon (1 mentions)
Streptomycin, by Sangon (1 mentions)
Murine tpo, by R&D Systems (1 mentions)
8 protocols using ficoll hypaque premium
1
Ficoll-Hypaque Blood Purification Protocol
2
Isolation and Culture of Immune Cells from Japanese Sea Bass
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MO/MΦ, lymphocytes, and neutrophils were separated from caudal vein blood of healthy Japanese sea bass according to a previously described method (Liu et al., 2018 (link)). Briefly, heparinized blood was collected, and cells were isolated following sedimentation with 6% dextran T 500 (Sigma, USA). After low-speed centrifugation at 400 g for 25 min at 24 °C, cells packed below Ficoll-Hypaque PREMIUM (GE Healthcare) (i. e., erythrocytes and neutrophils) were subjected to hypotonic lysis with ice-cold ACK (Ammonium-Chloride-Potassium) Lysis Buffer (0.15 mol/L NH4Cl, 0.01 mol/L KHCO3, 0.1 m mol/L EDTA) to eliminate red blood cells. The resulting neutrophil suspension was washed and suspended in RPMI 1640 medium (Invitrogen, China). The buffer layer above the Ficoll-Hypaque PREMIUM was collected and washed carefully, and the number of cells was determined using a hemocytometer (Sangon, China). Cells were cultured in 35 mm dishes for 12 h, and adherent MO/MΦ and non-adherent lymphocytes were carefully collected and cultured in complete medium (RPMI 1640 supplemented with 5% (v/v) Japanese sea bass serum, 5% (v/v) fetal bovine serum (FBS, Invitrogen), 100 U/mL penicillin, and 100 μg/mL streptomycin) at 24 °C with 5% CO2.
3
Erythrocyte Lysis Assay for Allograft Rejection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We employed erythrocyte lysis assay to investigate the allograft rejection as previously described57 (link). The PBMC were separated using Ficoll-Hypaque PREMIUM (1.077 g/ml) (GE Healthcare) in combination with centrifugation according to the manufacturer’s instructions. Ayu anti-CSF1R antibody was prepared in our lab58 . PBMC were incubated with primary ayu anti-CSF1R antibodies for 30 min at 4 °C. Cells were then incubated with goat-anti-mouse-Ig microbeads (Miltenyi Biotec, Germany) after washing twice. Unlabeled cells flowing through the column were collected as ayu PBL.
Ayu erythrocytes were prepared as target cells57 (link). To quantify the killing activity of alloreactive PBL, a hemoglobin release assay was carried out57 (link). In this assay effector cells are co-cultured with erythrocyte targets and hemoglobin released from killed erythrocytes into the supernatant is measured colorimetrically using 3,3′,5,5′-tetramethylbenzidine (TMB). The percentage of specific cytotoxicity was calculated using the following formula: % Specific cytotoxicity = [(EXP–ESR)–TSR]/[(TMR–VCC) - TSR] × 100%. EXP: experimental wells were filled with varying numbers of ayu PBL and a constant number of target erythrocytes, ESR: effector cell spontaneous release, TSR: target cell spontaneous release, TMR: target cell maximum release, VCC: volume correction control.
Ayu erythrocytes were prepared as target cells57 (link). To quantify the killing activity of alloreactive PBL, a hemoglobin release assay was carried out57 (link). In this assay effector cells are co-cultured with erythrocyte targets and hemoglobin released from killed erythrocytes into the supernatant is measured colorimetrically using 3,3′,5,5′-tetramethylbenzidine (TMB). The percentage of specific cytotoxicity was calculated using the following formula: % Specific cytotoxicity = [(EXP–ESR)–TSR]/[(TMR–VCC) - TSR] × 100%. EXP: experimental wells were filled with varying numbers of ayu PBL and a constant number of target erythrocytes, ESR: effector cell spontaneous release, TSR: target cell spontaneous release, TMR: target cell maximum release, VCC: volume correction control.
4
Enrichment of CD34+ Cells from Nonhuman Primate BM
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CD34+ cells were enriched from fresh total mononuclear cells (MNCs) of nonhuman primates with the magnetic-activated cell sorting (MACS) immunomagnetic absorption column separation device, coupled with mouse anti-primate CD34 antibody (BD, USA) and anti-mouse IgG MicroBead (Miltenyi Biotec, Germany), according to the manufacturer’s instructions. MNCs were obtained from fresh nonhuman primate BM using density centrifugation with Ficoll-Hypaque Premium (GE Healthcare, USA). The purity of CD34+ cells was verified using flow cytometry, with anti-primate CD34 mAb conjugated with phycoerythrin (PE; Immunotec, Canada) and the BD FACSVerse flow cytometer (BD, USA).
5
Isolation and Culture of PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For isolation of peripheral blood mononuclear cells (PBMCs), 10~20 ml of peripheral blood was collected by venipuncture donor. Cells were diluted by 2~4× volume of PBS and layered over 20 ml of Ficoll-Hypaque Premium (GE Healthcare) gradient, according to the manufacturer’s instructions, and centrifuged at 1200×g for 40 min. The mononuclear cell layer (containing PBMCs) was carefully transferred and washed with PBS for twice. PBMCs wereresuspended in blood medium (1: 1 Iscove's Modified Dulbecco's Medium with glutamine/F12, 5 mg/ml Human serum albumin, 1× lipid concentrate, 10 μg/ml insulin, 100 μg/ml transferrin, 14 ng/ml sodium selenite, 64 μg/ml L-ascorbic acid 2-phosphate, 450 μM 1- thioglycerol, 50 ng/ml SCF, 10 ng/ml IL3, 2 U/ml EPO, 40 ng/ml IGF1, and 1 μM dexamethasone, all bought from Invitrogen) to the density of two to three million cells per ml. Media were changed every 2 or 3 days.
6
Isolation and Culture of Kidney-Derived Macrophages from Mudskippers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Kidney-derived MO/MФ from mudskippers were isolated and cultured as reported previously (Guan et al., 2017 (link); Shen et al., 2020 (link)). Briefly, fish kidney leukocyte-enriched fractions were obtained using Ficoll-Hypaque PREMIUM (1.077 g/mL) (GE Healthcare, USA). Non-adherent cells were removed by washing, and adherent cells were incubated in complete medium (RPMI 1640, 5% mudskipper serum, 5% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, USA), 100 U/mL penicillin, 100 μg/mL streptomycin) at 24 °C with 5% CO2. The HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (Biochrom AG, Germany) supplemented with 10% (v/v) FBS, 100 U/mL penicillin (Sangon Biotech, China), and 100 μg/mL streptomycin (Sangon Biotech) at 37 °C with 5% CO2. The HEK293T cells were then seeded into 6-well plates to allow growth until 70%–90% confluence on the day of transfection.
7
Measuring Integrin Activation in Murine Bone Marrow Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bone marrow cells were harvested from 8- to 9-wk-old WT and kindlin-3 hypomorphic mice (Xu et al, 2014 (link); Meller et al, 2017 (link)) by flushing femurs with Catch Buffer (PBS pH 7.4, 2% BSA, 0.38% trisodium citrate, DNAse I (1 U/ml), penicillin/streptomycin (100 U/ml). Mononuclear cells were isolated by centrifugation over Ficoll Hypaque Premium (1.080 g/ml; GE Healthcare) at 400g for 30 min at 22°C. Low-density mononuclear cells were cultured for up to 5 d at a starting density of 1 × 106/ml in serum-free IMDM medium supplemented as described (Zauli et al, 1997 (link); Shiraga et al, 1999 (link)) in the presence of 50 ng/ml murine TPO (R&D Systems) and 10 ng/ml murine IL-11 and IL-6 (Millipore Sigma). After 5 d in culture, the cells were treated with lentiviral vectors (Xu et al, 2018 (link)) expressing EGFP-tagged kindlin-3 (WT), QW/AA, TS/AA, or EGFP only as described. 48 h post-transduction, the cells were either untreated or stimulated with PAR-4 agonist peptide (100 μg/ml; Bachem) for 30 min at 37°C. αIIbβ3 integrin activation was measured by flow cytometry of EGFP-positive cell populations using PE-labeled Ab to activation-dependent epitope of CD41/CD61 (clone JON/A) (Emfret Analytics) and rat antimouse total CD41-PerCP/Cy5.5 (BioLegend). The data are expressed as % activation = (MFI JONA/MFI total CD41) × 100.
8
Isolation and Culture of Mudskipper Myeloid Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mudskipper kidney-derived MOs/MФs were isolated and cultured as described previously (Ding et al., 2019 (link)). Briefly, fish kidney leukocyte-enriched fractions were obtained using Ficoll-Hypaque PREMIUM (1.077 g/mL; GE Healthcare, USA). After washing twice with RPMI1640, the MOs/MФs were cultured in 35 mm dishes at a concentration of 2×107 cells/mL. Cells (2 mL) were then incubated for at least 12 h at 24 °C with 5% CO2. Non-adherent cells were washed off, and adherent cells were incubated in complete medium (RPMI 1640, 5% mudskipper serum, 5% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin) at 24 °C with 5% CO2. The purity of the isolated mudskipper MOs/MФs was greater than 95%, as measured by Wright-Giemsa staining.
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!