Vero E6 cells (CRL-1586, American Type Culture Collection, ATCC, Manassas, VA) and Calu-3 cells (HTB-55, ATCC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific) supplemented with 20% v/v of filtered fetal bovine serum (FBS, Thermo Fisher Scientific), 1% v/v penicillin/streptomycin (Pen/Strep, Thermo Fisher Scientific) and 1% v/v of GlutaMAX supplement (Thermo Fisher Scientific). Human epithelial colorectal adenocarcinoma cell line Caco-2 (HTB-37, ATCC) was cultured in Minimum Essential Medium (Thermo Fisher Scientific) supplemented with 20% FBS, 1% Pen/Strep, and 1% GlutaMAX supplement. Primary human umbilical vein endothelial cells (HUVEC), pooled from multiple donors, were supplied by Invitrogen (Thermo Fisher Scientific) cryopreserved at the end of the primary culture stage. These cells were cultured in adhesion in Medium 200 (M200, Thermo Fisher Scientific), supplemented with 1% v/v Large Vessel Endothelial Supplement (LVES 50x, Thermo Fisher Scientific) and 1% v/v Pen/Strep. Peripheral blood mononuclear cells were purified by Ficoll-Paque PREMIUM (Merck) gradient from healthy blood donors and grown in RPMI 1640 Medium (Thermo Fisher Scientific) supplemented with 10% FBS, 1% Pen/Strep, 1% GlutaMAX and 1% Hepes. For the experiments, cells were seeded in 6-well or 12-well plates and maintained at 37°C in a humidified 5% CO2 incubator.
Ficoll paque premium
Manufactured by Merck Group
Sourced in United States
Ficoll-Paque PREMIUM is a density gradient medium used for the isolation and purification of cells, particularly lymphocytes, from whole blood or other biological samples. It is designed to provide a simple and efficient method for the separation of cell types based on their differential migration during centrifugation.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Facsaria 3, by BD (2 mentions)
Normal rabbit serum, by Thermo Fisher Scientific (2 mentions)
Live dead fixable aqua, by Thermo Fisher Scientific (2 mentions)
Strep tactin xt dy 488, by IBA Lifesciences (2 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Glutamax supplement, by Thermo Fisher Scientific (1 mentions)
Vero e6 cell, by American Type Culture Collection (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Calu 3 cells, by American Type Culture Collection (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Anti human cd14 magnetic particles, by BD (1 mentions)
Cd14 pe cy7, by BioLegend (1 mentions)
Cd56 pe cy7, by BioLegend (1 mentions)
Cd3 pe cy7, by BioLegend (1 mentions)
Igd a700, by BD (1 mentions)
Igm percp cy5, by BD (1 mentions)
Cd27 pe, by BD (1 mentions)
15 protocols using ficoll paque premium
1
Cell Culture Protocols for Diverse Cell Lines
2
Blood Smear Preparation and PBMC Isolation
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One drop (approximately 20 µl) of blood was used to prepare blood smears on Thermo Scientific™ SuperFrost Plus™ (Thermo Fisher Scientific, 15438060). Blood smears were air dried for at least 24 h prior to staining.
Serum was extracted for lectin blotting by centrifugation of EDTA blood for 10 min at 600 × g.
For isolation of peripheral blood mononuclear cells (PBMCs), EDTA blood was diluted 1:1 with phosphate‐buffered saline (PBS). 10 ml Ficoll®‐Paque Premium (Merck, 17‐5442‐02) was carefully overlayered with 20 ml of the 1:1 blood/PBS mixture in a 50 ml tube. The sample was centrifuged at 400 × g for 30 min at 20°C. The layer containing mononuclear cells was transferred into a 15‐ml tube. Cells were diluted with 10 ml PBS and centrifuged at 400 × g for 10 min at 20°C. The supernatant was discarded, and the pellet was suspended in 1 ml PBS.
Serum was extracted for lectin blotting by centrifugation of EDTA blood for 10 min at 600 × g.
For isolation of peripheral blood mononuclear cells (PBMCs), EDTA blood was diluted 1:1 with phosphate‐buffered saline (PBS). 10 ml Ficoll®‐Paque Premium (Merck, 17‐5442‐02) was carefully overlayered with 20 ml of the 1:1 blood/PBS mixture in a 50 ml tube. The sample was centrifuged at 400 × g for 30 min at 20°C. The layer containing mononuclear cells was transferred into a 15‐ml tube. Cells were diluted with 10 ml PBS and centrifuged at 400 × g for 10 min at 20°C. The supernatant was discarded, and the pellet was suspended in 1 ml PBS.
3
Isolation and Purification of CD14+ Monocytes from Peripheral Blood
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Venous blood samples were obtained from the controls and the study group. Peripheral blood mononuclear cells (PBMCs) were separated from whole blood by density gradient centrifugation using Ficoll–Paque Premium (17-5442-02, Merck, Kenilworth, New Jersey, U.S.A.). Specifically, 4 mL of Ficoll solution was added to the bottom of a 15 mL tube before the addition of 8 mL of whole blood. After centrifugation at 400 g for 40 min at 25 °C, PBMCs were collected from the plasma/Ficoll interface.
After PBMC separation, CD14+ cells were isolated using anti-human CD14 magnetic particles (557769, BD Bioscience, Franklin Lakes, New Jersey, U.S.A.). PBMCs were diluted with equal amounts of phosphate-buffered saline and centrifuged at 240 g for 5 min. The supernatant was discarded, and the cell pellets were resuspended in 1X BD IMag™ buffer. Cell numbers were determined, and cell suspensions were centrifuged at 200 g for 10 min. For every 107 cells, 50 μL of BD IMag™ anti-human CD14 magnetic particles was added to the supernatant. The cell-magnetic particle mixture was incubated at room temperature for 30 min. After washing with 1X BD IMag™ buffer, pellets of CD14+ cells were collected and resuspended in 500 μL of QIAzol lysis reagent (RLT buffer, RNeasy Mini Kit, Qiagen, Venlo, Netherlands) for further use.
After PBMC separation, CD14+ cells were isolated using anti-human CD14 magnetic particles (557769, BD Bioscience, Franklin Lakes, New Jersey, U.S.A.). PBMCs were diluted with equal amounts of phosphate-buffered saline and centrifuged at 240 g for 5 min. The supernatant was discarded, and the cell pellets were resuspended in 1X BD IMag™ buffer. Cell numbers were determined, and cell suspensions were centrifuged at 200 g for 10 min. For every 107 cells, 50 μL of BD IMag™ anti-human CD14 magnetic particles was added to the supernatant. The cell-magnetic particle mixture was incubated at room temperature for 30 min. After washing with 1X BD IMag™ buffer, pellets of CD14+ cells were collected and resuspended in 500 μL of QIAzol lysis reagent (RLT buffer, RNeasy Mini Kit, Qiagen, Venlo, Netherlands) for further use.
4
Isolation and Single-Cell Sorting of SARS-CoV-2 Reactive Memory B Cells
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Peripheral blood mononuclear cells (PBMCs) and the single-cell sorting strategy were performed as previously described5 (link). In brief, PBMCs were isolated from heparin-treated whole blood by density gradient centrifugation (Ficoll-Paque PREMIUM, Sigma-Aldrich). After separation, PBMCs were stained with Live/Dead Fixable Aqua (Invitrogen; Thermo Scientific) diluted 1:500 at room temperature. After 20 min of incubation, cells were washed with PBS and unspecific bindings were saturated with 20% normal rabbit serum (Life Technologies). Following 20 min of incubation at 4 °C, cells were washed with PBS and stained with SARS-CoV-2 S protein labelled with Strep-Tactin XT DY-488 (2-1562-050, iba-lifesciences) for 30 min at 4 °C. After incubation, the following staining mix was used CD19 V421 (1:320; 562440, BD), IgM PerCP-Cy5.5 (1:50; 561285, BD), CD27 PE (1:30; 340425, BD), IgD-A700 (1:15; 561302, BD), CD3 PE-Cy7 (1:100; 300420, BioLegend), CD14 PE-Cy7 (1:320; 301814, BioLegend), CD56 PE-Cy7 (1:80; 318318, BioLegend) and cells were incubated at 4 °C for an additional 30 min. Stained memory B cells were single-cell-sorted with a BD FACS Aria III (BD Biosciences) into 384-well plates containing 3T3-CD40L feeder cells and were incubated with IL-2 and IL-21 for 14 days as previously described25 (link).
5
Isolation and Expansion of SARS-CoV-2-Specific B Cells
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Blood samples were screened for SARS-CoV-2 RNA and for antibodies against HIV, HBV and HCV. Peripheral blood mononuclear cells (PBMCs) were isolated from heparin-treated whole blood by density gradient centrifugation (Ficoll-Paque PREMIUM, Sigma-Aldrich). After separation, PBMC were stained with Live/Dead Fixable Aqua (Invitrogen; Thermo Scientific) in 100 μL final volume diluted 1:500 at room temperature RT. After 20 min incubation cells were washed with PBS and unspecific bindings were saturated with 50 μL of 20% normal rabbit serum (Life technologies) in PBS . Following 20 min incubation at 4°C cells were washed with PBS and stained with SARS-CoV-2 S-protein labeled with Strep-Tactin®XT DY-488 (iba-lifesciences cat# 2-1562-050) for 30 min at 4°C. After incubation the following staining mix was used CD19 V421 (BD cat# 562440), IgM PerCP-Cy5.5 (BD cat# 561285), CD27 PE (BD cat# 340425), IgD-A700 (BD cat# 561302), CD3 PE-Cy7 (BioLegend cat# 300420), CD14 PE-Cy7 (BioLegend cat# 301814), CD56 PE-Cy7 (BioLegend cat# 318318) and cells were incubated at 4°C for additional 30 min. Stained MBCs were single cell-sorted with a BD FACS Aria III (BD Biosciences) into 384-well plates containing 3T3-CD40L feeder cells and were incubated with IL-2 and IL-21 for 14 days as previously described (Huang et al., 2013 (link)).
6
Isolation of Mononuclear Cells from Blood
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Blood samples were obtained from the Hematology and Oncology Department of Shanghai Children's Medical Center, and patient’s mother provided informed consent. MNCs were isolated from PB samples using standard Ficoll procedures; 8 ml of diluted blood (blood: PBS = 1:2) was loaded onto a 3 ml layer of Ficoll-Paque PREMIUM (p = 1.077 g/ml; Sigma) in a 15-ml conical tube.
7
Menstrual Blood-Derived Stem Cell Isolation
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Menstrual blood samples were collected with a DivaCup during the second and third days of menstruation. Once received in the laboratory, menstrual blood samples were transferred into a 50 mL tube and treated with 50 U/mL collagenase II (Sigma-Aldrich, St. Louis, MO, USA) for 20 min at 37 °C in a humidified 5% CO2. MenSC isolation was immediately performed using the “Ficoll-Paque™ premium” (Sigma-Aldrich, St. Louis, MO, USA) according to manufacturer’s protocol. After isolation MenSC washed in phosphate-buffered saline (PBS) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) twice and suspended in growth medium DMEM/F12 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin (100 U/mL)—streptomycin (100 µg/mL) solution (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and, then, seeded into a 75 cm2 plastic cell culture flasks and cultivated at 37 °C in a humidified 5% CO2 atmosphere. After 3–5 days of culture, non-adherent cells were washed away with PBS (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) leaving behind adherent fibroblastic cells growing in clusters. The growth medium was replaced every 3 days.
8
Isolation and Culture of Human Monocytes
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Leukocyte concentrates (buffy coats) from healthy donor volunteers were obtained from the Blood Transfusion Service at Massachusetts General Hospital with approval from the institutional review board (IRB protocol no. 2019P000585). Peripheral blood mononuclear cells were freshly isolated from the buffy coats by Ficoll-Paque PREMIUM (1.073 g/mL, Sigma-Aldrich, St. Louis, MO, USA) followed by erythrocyte lysis using an ice-cold NH4Cl solution for 10 min. Monocyte (CD14+CD16−) negative selection was performed via magnetic-activated cell sorting using the Classical Monocyte Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. The human monocytes were flushed with PBS containing 2.5% human serum albumin (HSA) and 2.5 mM EDTA. This yielded >75% monocytes, as determined by flow cytometry following labeling with anti-CD14-FITC mAb (BD Biosciences, San Jose, CA, USA).
The human THP-1 monocytic cell line was cultured in RPMI 1640 medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 50 μM β-mercaptoethanol and 100 units/mL penicillin-streptomycin (Invitrogen, Waltham, MA, USA). Cells were plated and maintained at a density of 2.5 × 105 cells/mL by evaluating the number of cells every 2 days.
The human THP-1 monocytic cell line was cultured in RPMI 1640 medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 50 μM β-mercaptoethanol and 100 units/mL penicillin-streptomycin (Invitrogen, Waltham, MA, USA). Cells were plated and maintained at a density of 2.5 × 105 cells/mL by evaluating the number of cells every 2 days.
9
Efficient Isolation of PB MNCs
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All human whole blood samples were obtained from volunteers at the Institute of Hematology and Blood Diseases Hospital. Each PB sample was divided equally into four parts. MNCs were isolated from part 1 using standard Ficoll procedures by loading 35 ml of diluted blood (blood:PBS = 1:2) onto a 15-ml layer of Ficoll-Paque PREMIUM (p = 1.077 g/ml; Sigma Aldrich) in a 50-ml conical tube. MNCs were isolated from part 2 using red cell lysis buffer procedures, with the addition of 4-fold of ACK buffer to the blood and centrifugation after 20 min of incubation in a 37 °C water bath. Hydroxyethyl starch (HES; Sigma Aldrich) of 60-kDa molecular mass was added to PB from parts 3 and 4 in a 1:5 ratio. The supernatants collected after the sample remained stationary for 40 min at room temperature and were processed to yield MNCs either by the Ficoll method for part 3 or by the ACK method for part 4. In total, we used two isolating methods for enrichment of PB MNCs, including Ficoll and HES-Ficoll, and two isolating methods without enrichment, including ACK and HES-ACK. Except for MNCs, the other types of white blood cells would die quickly after being cultured in vitro, so we designated the isolated cells PB MNCs.
10
Isolation and Reprogramming of Human iPSCs from PBMCs
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The peripheral blood mononuclear cells (PBMCs) were isolated from the blood samples using Ficoll-Paque Premium according to the manufacturer's instructions (Sigma-Aldrich). The PBMCs were cultured in StemPro-34 SFM Complete Medium (Gibco) for at least 4 days before reprogramming. The cells were transduced with the CytoTune-iPS 2.0 Sendai reprogramming kit (Thermo Fisher Scientific). At day 3 after reprogramming, the cells were seeded on Matrigel-coated plates and cultured in StemPro medium without cytokines. At day 7, half of the medium was replaced with ReproTeSR medium (Stem Cell Technologies) and the medium was completely changed to ReproTeSR at day 8. The iPSCs colonies generated between days 15 and 30 were manually picked, expanded, and maintained in mTESR-1 medium (Stem Cell Technologies) (Fig. 1A ). The established hiPSC lines were registered in (https://hpscreg.eu ) website and were assigned unique stem cell line names as follows: QBRIi001-A (Ctr1-iPSCs-C1), QBRIi001-B (Ctr1-iPSCs-C2), QBRIi001-C (Ctr1-iPSCs-C3), QBRIi002-A (Ctr2-iPSCs-C1), QBRIi002-B (Ctr2-iPSCs-C2), QBRIi002-C (Ctr2-iPSCs-C3), QBRIi005-A (PsO1-iPSCs-C1), QBRIi005-B (PsO1-iPSCs-C2), QBRIi005-C (PsO1-iPSCs-C3), QBRIi006-A (PsO2-iPSCs-C1), QBRIi006-B (PsO2-iPSCs-C2), and QBRIi006-C (PsO2-iPSCs-C3).
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