Sorted LT-HSCs, ST-HSCs or MEPs were cultured for 36–48 h in serum-free X-VIVO 10 (Lonza) media with 1% Bovine Serum Albumin Fraction V (Roche, 10735086001), 1× l -Glutamine (Thermo Fisher, 25030081), 1× Penicillin–Streptomycin (Thermo Fisher, 15140122) and the following cytokines (all from Miltenyi Biotec): FLT3L (100 ng/mL), G-CSF (10 ng/mL), SCF (100 ng/mL), TPO (15 ng/mL), and IL-6 (10 ng/mL). Cells were cultured in 96-well U-bottom plates (Corning, 351177).
Bovine serum albumin fraction 5
Manufactured by Roche
Sourced in Germany, United States, Switzerland, Belgium
Bovine serum albumin fraction V is a highly purified protein derived from bovine blood serum. It is commonly used as a reagent in various laboratory procedures and assays, serving as a stabilizer, blocking agent, or protein source.
Automatically generated - may contain errors
Lab products found in correlation
Heparin, by Leo pharma (7 mentions)
Ko143, by Bio-Techne (6 mentions)
Nystatin, by Merck Group (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Fibronectin, by Merck Group (3 mentions)
Bovine serum albumin fraction 5 fatty acid free, by Roche (2 mentions)
Advanced dmem f12 medium, by Thermo Fisher Scientific (2 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
Gentamicin, by Merck & Co (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (2 mentions)
Tpck treated trypsin, by Merck Group (2 mentions)
X vivo 10, by Lonza (1 mentions)
Flt3l, by Miltenyi Biotec (1 mentions)
G csf, by Miltenyi Biotec (1 mentions)
U bottom plate, by Corning (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Millex gv 0.22 μm filter, by Merck Group (1 mentions)
42 protocols using bovine serum albumin fraction 5
1
Ex Vivo Expansion of Hematopoietic Stem Cells
2
Conjugation of Gold Sulfo-Tag NHS-Ester
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All chemicals were used as received without further purification and all aqueous solutions were prepared with MQ water. GOLD SULFO-TAG NHS-Ester lyophilized (ref. RA19AO), read buffer T 4X (ref. R92TC), conjugation buffer (ref. R60AJ), and conjugation storage buffer (ref. R60AC) were purchased from Meso Scale Discovery (MSD). Other materials included: Zeba Spin desalting columns 40 K MWCO 0.5 mL (ref. 87766, ThermoFisher, Waltham, MA, USA), Millex-GV Filter 0.22 μm (SLGV004SL, Sigma-Aldrich, MO, USA), syringe 1 mL BD Luer-Lok tip (ref. 309628, BD, New York, NJ, USA), Trizma base (ref. 1002134476, Sigma-Aldrich, MO, USA), bovine serum albumin fraction V (ref. 10735078001, Roche Diagnostics, Rotkreuz, Switzerland), glycerol (ref. 49770-250 mL, Sigma-Aldrich, MO, USA), PBS 10x pH 7.4 phosphate saline buffer (ref. 7011-044, Gibco, Billings, MT, USA), Tween-20 (ref. P1379-100 mL, Sigma-Aldrich, MO, USA), CaCl2·2H2O (ref. 223506, Fluka, Buchs, Switzerland). Technical grade ethanol was used for cleaning the SPCE. The FABP free human serum was provided by HyTest (ref. 8FFS, HyTest Ltd., Turku, Finland). The GFAP and S100β levels in this serum are negligible.
3
Measuring Viral Titers and Cytokines in Mouse Lungs
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At pre-inoculation and 3 or 5 days post-inoculation (dpi), mouse lungs were collected after euthanasia (n = 6). Lungs were homogenized with 2 mL of transport medium: MEM containing 10,000 U/mL penicillin G, 10 mg/mL streptomycin, 0.3 mg/mL gentamicin, 250 U/mL Nystatin (Sigma-Aldrich, St. Louis, MO, USA), and 0.5% bovine serum albumin fraction V (Roche, Basel, Switzerland). The homogenized lung tissue was centrifuged for 5 min at 4 °C and 8000 rpm. The supernatant was collected, and all samples were stored at −80 °C until quantification of virus titers and cytokines.
4
Enrichment of LRP6-Expressing Cells
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Cells (10 × 107) were pelleted and resuspended in 500 μL of PBS supplemented with 0.5% BSA (Bovine Serum Albumin Fraction V, Roche) and 2 mM EDTA. An anti-LRP6 antibody (C10, Santa Cruz Biotechnology) was added to the cell suspension and incubated on a rotating wheel at 4°C for 1 h. The cells were then enriched using anti-mouse IgG Microbeads (Miltenyi Biotech) and LS columns (Miltenyi Biotec), according to the manufacturer's instructions. In order to further increase the percentage of LRP6-expressing cells, similar procedures were repeated twice.
5
Biotinylated Peptide/Protein ELISA
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Example 9
Elisa
To not-coated Maxisorb plates non-biotinylated peptide/protein/aggregate and to streptavidin coated Maxisorb plates biotinylated peptide/protein/aggregate in PBS were added and incubated over-night. The supernatant was discarded and the wells washed three times with 90 μl wash buffer (1×PBS/0.1% Tween 20). Remaining reactive spots were blocked with blocking buffer (1×PBS/2% BSA (Bovine Serum Albumin Fraction V. fatty acid free, Roche, Cat. No.: 10735078001)10.05% Tween 20) by incubating for 1 h. The supernatant was discarded and the wells washed three times with 90 μl wash buffer. Samples and control antibody were prepared in 12 dilutions. (1:2) in ELISA buffer (1×PBS/0.5% BSA (Bovine Serum Albumin Fraction V, fatty acid free, Roche, Cat. No.: 10735078001)/0.05% Tween 20) with a start concentration of 500 ng/mL. The incubation time was 60 minutes at RT on a shaker. The supernatant was discarded and the wells washed three times with 90 μl wash buffer. Solutions of the secondary antibody were prepared in ELISA buffer. A total of 25 μl antibody-mix was transferred in all wells of the assay plate and the plate was thereafter incubated on shaker for 60 minutes at RT. The supernatant was discarded and the wells were washed three times with 90 μl wash buffer. To all wells 25 μl of ABTS solution was added. The absorbance was read at 405 nm-492 nm.
6
Immortalized Human Pancreatic Cell Line
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A fragment from neonatal pancreas was collected, cut into pieces, digested with type IV collagenase (Sigma-Aldrich) and transduced with lentiviral vectors. Two loxP sites flank the integrated sequences expressing SV40T and hTERT, allowing subsequent excision dependent on Cre recombinase expression15 (link). The tissue was next transplanted under the kidney capsule of immune-incompetent SCID (Charles River, L’Arbresle, France) as described49 (link). Following 3 successive rounds of transplantation, we derived the cell line ECN90 that is cultured at 37 °C in 5% CO2 in Advanced DMEM/F12 medium (Thermo Fisher Scientific) supplemented with 2% bovine serum albumin fraction V (Roche), 6.7 ng/ml sodium selenite, 10 mM nicotinamide (Calbiochem), 50 μM β-mercaptoethanol (Sigma-Aldrich) and penicillin/streptomycin (Thermo Fisher Scientific).
7
Biotinylated Peptide/Protein ELISA
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Example 9
Elisa
To not-coated Maxisorb plates non-biotinylated peptide/protein/aggregate and to streptavidin coated Maxisorb plates biotinylated peptide/protein/aggregate in PBS were added and incubated over-night. The supernatant was discarded and the wells washed three times with 90 μl wash buffer (1×PBS/0.1% Tween 20). Remaining reactive spots were blocked with blocking buffer (1×PBS/2% BSA (Bovine Serum Albumin Fraction V. fatty acid free, Roche, Cat. No.: 10735078001)10.05% Tween 20) by incubating for 1 h. The supernatant was discarded and the wells washed three times with 90 μl wash buffer. Samples and control antibody were prepared in 12 dilutions. (1:2) in ELISA buffer (1×PBS/0.5% BSA (Bovine Serum Albumin Fraction V, fatty acid free, Roche, Cat. No.: 10735078001)/0.05% Tween 20) with a start concentration of 500 ng/mL. The incubation time was 60 minutes at RT on a shaker. The supernatant was discarded and the wells washed three times with 90 μl wash buffer. Solutions of the secondary antibody were prepared in ELISA buffer. A total of 25 μl antibody-mix was transferred in all wells of the assay plate and the plate was thereafter incubated on shaker for 60 minutes at RT. The supernatant was discarded and the wells were washed three times with 90 μl wash buffer. To all wells 25 μl of ABTS solution was added. The absorbance was read at 405 nm-492 nm.
8
CSFV Titration and Immunostaining
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SK-L cells were inoculated with the serially 10-fold diluted CSFV in 96-well plates and incubated at 37 °C for 72 to 96 h. Then, the plates were immunostained as described previously [27 (link)]. Briefly, the plate was air-dried and heat-fixed at 80 °C for 1 h. Cells were then incubated at room temperature for 1 h in the presence of the primary monoclonal antibody for NS3, 46/1 [28 (link)]. After washing with PBS, cells were incubated at 37 °C for 1 h in the presence of goat anti-mouse IgG (H + L) horseradish peroxidase conjugate (Bio-Rad, Hercules, CA, USA). The primary and secondary antibodies were diluted 2000-fold with PBS containing 1% Bovine Serum Albumin Fraction V (Roche, Basel, Switzerland) and 0.05% Tween 20 (FUJIFILM Wako Pure Chemical Corp.). Finally, cells were washed again with PBS and then stained with 3-amino-9-ethyl carbazole (Sigma-Aldrich). Virus titers were calculated and expressed as TCID50 per milliliter [29 (link)].
9
Culturing Human Pancreatic β Cells
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The human pancreatic β cell line ECN9035 (link) (HLA-I haplotype: HLA-A∗02:01/03:01, -B∗40:01/49:01, -C∗03:04/07:01) was cultured on plates coated with 1.2% Matrigel and 3 μg/ml fibronectin (both from Sigma-Aldrich) at 37 °C in 5% CO2 in Advanced DMEM/F12 medium (Thermo Fisher Scientific) supplemented with 2% bovine serum albumin fraction V (Roche), 6.7 ng/ml sodium selenite, 10 mM nicotinamide (Calbiochem), 50 μM β-mercaptoethanol (Sigma-Aldrich), 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific). Experiments were carried out 48 h after seeding the cells at a density of 105 cells/cm2. Advanced DMEM/F12 medium depleted in either glutamate (Thermo Fisher Scientific) or tryptophan (MyBioSource) was used in some experiments.
10
Cultivation of EndoC-βH1 Cells
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EndoC-βH1 cells (Human Cell Design, France) [32 (link)] were grown in DMEM (low glucose, pyruvate) supplemented with 2% wt/vol Bovine Serum Albumin Fraction V, fatty acid free (Roche, USA/UK), 50 μmol/l 2-mercaptoethanol, 10 nmol/l nicotinamide (Sigma-Aldrich, USA/UK), 5.5 μg/ml transferrin (Sigma-Aldrich, USA/UK), 6.6 ng/ml sodium selenite (Sigma-Aldrich, USA/UK), 100 U/ml penicillin and 100 µg/ml streptomycin and 2 mmol/l l -glutamine on cell culture plates coated with DMEM (high glucose) supplemented with 1% vol/vol extracellular matrix (Sigma-Aldrich, USA/UK), 2 μg/ml fibronectin (Sigma-Aldrich, USA/UK) and 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C and 5% CO2. Cell culture reagents were manufactured by ThermoFisher Scientific (USA/UK) unless otherwise stated. All EndoC-βH1 cell lines were routinely tested and were negative for mycoplasma.
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