Housing and care of the animals were in accordance with a protocol approved by the Institutional Animal Care and Use Committee. Eight- to 12-week-old female NOD/LtSz-IL2R γ–c (NSG) mice (Jackson Lab, Sacramento, CA; strain no. 005557) were used for human CD34+ transplantation studies. Twenty-four hours prior to transplantation, mice were injected intraperitoneally with 35 mg/kg busulfan (BUSULFEX; PDL BioPharma, Redwood City, CA). Twelve to 13 weeks post-transplantation, NSG mice were sacrificed and bone marrow cells were collected from tibias and femurs in Dulbecco’s phosphate-buffered saline (DPBS) (Corning; CellGro, Herndon, VA; cat. no. 21-013-CV), supplemented with 2% heat inactivated fetal calf serum (HI-FCS) (Thermo Scientific; HyClone, Logan, UT; cat. no. SH30396.03). Six- to 8-week-old female WAS–/– and male WAS– mice derived on a C57Bl/6 strain as described previously41 (link) were used to compare strength of WASp expression using different promoters.
Fcs hi
Manufactured by Thermo Fisher Scientific
Sourced in Italy
The FCS hi is a flow cytometry instrument designed for sensitive and accurate detection of fluorescent signals. It is capable of measuring physical and fluorescent characteristics of cells or particles in a fluid stream.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640, by Thermo Fisher Scientific (6 mentions)
α mem, by Thermo Fisher Scientific (4 mentions)
B 27 minus insulin, by Thermo Fisher Scientific (3 mentions)
Chir99021, by Selleck Chemicals (3 mentions)
Endothelial cell basal medium 2, by PromoCell (3 mentions)
Sb431542, by Merck Group (3 mentions)
Y 27632, by Merck Group (3 mentions)
Pen strep, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Human fgfb, by R&D Systems (2 mentions)
Tryple express, by Thermo Fisher Scientific (2 mentions)
Rhesus il 2, by MyBioSource (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Vascular endothelial growth factor (vegf), by Merck Group (2 mentions)
Human il 2, by Thermo Fisher Scientific (2 mentions)
Human m csf, by Thermo Fisher Scientific (2 mentions)
Dulbecco s phosphate buffered saline dpbs, by Corning (1 mentions)
16 protocols using fcs hi
1
NSG Mice Xenograft and WAS Mice Comparison
2
PBMC Isolation and Immune Stimulation
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Peripheral blood mononuclear cells (PBMCs) were separated by density gradient over Ficoll-Paque (GE Healthcare) as previously described (36 (link)). PBMCs from patients with AP and AAH and corresponding controls were cryopreserved in heat inactivated fetal bovine serum (HI-FCS [ThermoFisher Scientific]), 10% dimethyl sulfide (DMSO [Santa Cruz Biotechnology]). All other experiments used freshly isolated PBMCs with no cryopreservation. Viability of all PBMCs were assessed prior to all experiments using trypan blue (Sigma Aldrich) and only those with viability over 70% for cryopreserved samples and 90% for fresh samples, were included. Cells were seeded at 1 million per mL in RPMI 1640 containing L-glutamine (Lonza), penicillin/streptomycin (Sigma Aldrich), and 10% HI-FCS. PBMCs were stimulated with 20 ng/mL lipopolysaccharide from Escherichia coli O55:B5 (LPS [Sigma-Aldrich]), 10 μg/mL polyinosilic:polycytidylic acid (poly I:C [Sigma-Aldrich]), 100 ng/mL flagellin from Salmonella typhimurium (Source BioScience), 1 μg/mL high mobility group box 1 protein (HMGB1 [Sigma-Aldrich]), 1,000 units/mL interferon alpha (IFNα [Peprotech]) or 50 ng/mL interleukin 1 alpha (IL-1α [R&D Systems]) for 3 or 24 h in 12 well plates, with 1 mL per well. Monocytes were magnetically isolated using the Pan monocyte isolation kit (Miltenyi Biotec) according to the manufacturer's instructions.
3
Preosteoblasts Proliferation on 3D Printed PLC+β-TCP
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The MC3T3-E1 preosteoblasts were plated at a density of 5000 cells/cm2 onto the sterilised 3D printed PLC+β-TCP 20% substrates which were contained in 48 well culture plates (Euroclone, SpA, Milano, Italy). The cells were grown for three days to an 80% confluence in α-MEM (Life Technologies, Monza, Italy) and supplemented with 10% HIFCS (Life Technologies, Monza, Italy), penicillin, and streptomycin (Sigma Aldrich, Milan, Italy). The control cultures were produced by growing cells on culture plates as substrates. Then, the cells were incubated with a CellTiter 96 Aqueous One Solution Reagent (Promega Italia Srl, Milan, Italy) for 2 h in a humidified 5% CO2 atmosphere. The coloured formazan was measured by reading the absorbance at 490 nm using a Tecan fluorometer (Tecan Italia Srl, Cernusco Sul Naviglio, Milan, Italy).
4
Mouse Osteoblast Cell Culture
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The MC3T3-E1 cell line utilized in the current study (mouse calvarian pre-osteoblasts), (ATCC, LGC Standards S.r.L Milano, Italy) were grown in Minimum Essential Medium Eagle (αMEM) (Life Technologies Milano, Italy) supplemented with 10% heat inactivated fetal calf serum (HI-FCS) (Life Technologies Milano, Italy) penicillin (100 U/ml), and streptomycin (50 μg/ml) (Life Technologies Milano, Italy).
5
Osteoblast Attachment and ECM Analysis on 3D Printed Scaffolds
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The MC3T3-E1 pre-osteoblasts were placed at a density of 5000 cells/cm2 into 12 well culture plates (Euroclone, SpA, Milano, Italy) and onto the previously sterilised 3D printed PCL+β-TCP 20% substrates. The cells were grown for four days in a minimum essential medium (α-MEM; Life Technologies, Monza, Italy) which was supplemented with 10% heat-inactivated fetal calf serum (HIFCS) (Life Technologies, Monza, Italy), penicillin, and streptomycin (Sigma Aldrich, Milano, Italy). Control cultures were produced by growing the cells on culture plates as substrates.
Then, all cultures were washed three times with phosphate-buffered saline (PBS) 0.1 M, pH 7.4 and fixed in 4% paraformaldehyde (PFA) which was diluted in PBS for 20 min at room temperature. The cultures were then washed three times with PBS, and the cells were stained with haematoxylin (5 min) and eosin (30 s). Following Dellinger et al. [43 (link)], 5% toluidine blue (5 s) was used to point out the extracellular matrix (ECM). After washing with tap water, the substrates were mounted on slides and imaged using a light microscope (Axiophot, Zeiss, Oberkochen, Germany). Image analysis was performed using NIH ImageJ software [44 (link)].
Then, all cultures were washed three times with phosphate-buffered saline (PBS) 0.1 M, pH 7.4 and fixed in 4% paraformaldehyde (PFA) which was diluted in PBS for 20 min at room temperature. The cultures were then washed three times with PBS, and the cells were stained with haematoxylin (5 min) and eosin (30 s). Following Dellinger et al. [43 (link)], 5% toluidine blue (5 s) was used to point out the extracellular matrix (ECM). After washing with tap water, the substrates were mounted on slides and imaged using a light microscope (Axiophot, Zeiss, Oberkochen, Germany). Image analysis was performed using NIH ImageJ software [44 (link)].
6
Mouse Calvaria Pre-Osteoblast Cell Culture
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The MC3T3-E1 cell line (mouse calvaria pre-osteoblasts) (ATCC, LGC Standards S.r.L Milano, Italy) was utilised in the current study. Cells were grown in minimum essential medium Eagle (αMEM) (Life Technologies, Milano, Italy) supplemented with 10% heat-inactivated fetal calf serum (HI-FCS) (Life Technologies, Milano, Italy), penicillin (100 U/mL), and streptomycin (50 µg/mL) (Life Technologies, Milano, Italy). Cells were detached using 0.25% trypsin for 2 min at room temperature and plated for each experimental protocol as below specified. The culture medium was replaced every 3 days.
7
ARPE-19 and NHDF Fibroblast Culture
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ARPE-19 cells (CRL-2302, ATCC) and neonatal NHDF fibroblasts (CC-2509, Lonza) were maintained and propagated in DMEM:F12 with L-glutamine (Biowhittaker) and MEM (Life Technologies) respectively, containing 10% heat-inactivated FCS (HI-FCS; Life Technologies) and 0.04% gentamicine. All infection experiments were done in 24-well plates (Costar).
8
Differentiation of iPSCs into Endothelial Cells
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Differentiation was initiated with B2M−/−CIITA−/− rhCD47 tg hiPSC at 60% confluency, and medium was changed to RPMI-1640 containing 2% B-27 minus insulin (both Gibco) and 5 µM CHIR-99021 (Selleckchem). On day 2, the medium was changed to reduced medium: RPMI-1640 containing 2% B-27 minus insulin (Gibco) and 2 µM CHIR-99021 (Selleckchem). From days 4–7, cells were cultured in RPMI-1640 EC medium of RPMI-1640 containing 2% B-27 minus insulin plus 50 ng/ml human VEGF, 10 ng/ml human FGFb; R&D Systems), 10 µM Y-27632 (Sigma-Aldrich), and 1 µM SB 431542 (Sigma-Aldrich). Endothelial cell clusters were visible from day 7 and cells were maintained in Endothelial Cell Basal Medium 2 (PromoCell) plus 10% FCS hi (Gibco), 1% pen/strep, 25 ng/ml VEGF, 2 ng/ml FGFb, 10 µM Y-27632 (Sigma-Aldrich), and 1 µM SB 431542 (Sigma-Aldrich). The differentiation process was completed after 14 d and undifferentiated cells detached during the differentiation process. TrypLE Express (Gibco) was used for passaging the cells 1:3 every 3–4 d.
9
Differentiation of Macrophages from PBMCs
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PBMCs were isolated by Ficoll separation from fresh blood and resuspended in RPMI-1640 with 10% FCS hi and 1% pen/strep (all from Gibco). Cells were plated in 24-well plates at a concentration of 106 cells/ml and 10 ng/ml rhesus M-CSF (Biorhyt). Medium was changed every second day until day 6. Macrophages were stimulated from day 6 with 1 µg/ml rhesus IL-2 (MyBioSource) for 24 h before the cells were used for assays.
10
Macrophage Differentiation from PBMCs
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PBMCs were isolated by Ficoll separation from fresh blood and resuspended in RPMI 1640 with 10% heat-inactivated FCS hi and 1% penicillin–streptomycin (all Gibco). Cells were plated in 24-well plates at a concentration of 1 × 106 cells per milliliter, and 10 ng ml−1 rhesus M-CSF (Biorbyt) was added. Media was changed every other day until day 6. Macrophages were stimulated from day 6 with 1 μg ml−1 rhesus IL-2 (MyBiosource) for 24 h before the cells were used.
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