A leuko-reduction collar was obtained from the Brigham and Women’s Hospital Crimson Core with patient information deidentified. All methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were reviewed and approved as exempt by the Harvard Faculty of Medicine Institutional Review Board. Primary human B cells were isolated from fresh leuko-reduction collar blood by using 750 µL of RosetteSep Human B Cell Enrichment Cocktail (Stemcell Technologies) for 10 mL of collar blood. The RosetteSep cocktail was incubated with collar blood for 20 min at room temperature, and then 10 mL of PBS supplemented with 2% FBS was added to the collar blood and mixed gently. The diluted collar blood was then layered on top of 10 mL Lymphoprep density gradient medium (Stemcell Technologies). After centrifugation at 1200 g for 20 min, the mononuclear cell layer was harvested and washed twice with PBS supplemented with 2% FBS. Purified cells were then resuspended in warm BD Quantum Yield medium (BD Biosciences) supplemented with 10% FBS and 1:50 Nutridoma-SP (Sigma Aldrich) at 106 cells/mL.
Nutridoma sp
Manufactured by Merck Group
Sourced in United States
Nutridoma-SP is a serum-free, protein-containing medium supplement designed for cell culture applications. It provides a source of growth-promoting factors and other essential nutrients to support cell growth and proliferation in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Quantum yield medium, by BD (2 mentions)
Hitrap protein g hp column, by GE Healthcare (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Rosettesep human b cell enrichment cocktail, by STEMCELL (1 mentions)
Lymphoprep density gradient medium, by STEMCELL (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Polyethylenimine (pei), by Merck Group (1 mentions)
0.22 μm filter, by Merck Group (1 mentions)
Pfuse higg1 fc2, by InvivoGen (1 mentions)
6 protocols using nutridoma sp
1
Isolation of Primary Human B Cells
2
Stimulation and Analysis of Human Lymphocytes
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Purified cells were resuspended in warm BD Quantum Yield medium supplemented with 10% FBS and 1:50 Nutridoma-SP (Sigma Aldrich) at 106 cells/mL and allowed to rest for 30 min at 37°C and 5% CO2. Cells were then pipeted several times to disperse clumps and were split into 96 wells for stimulation assays (100 µL per well). Cells were allowed to rest for 1 hr at 37°C and 5% CO2. Either 100 µL of medium (unstimulated condition) or 100 µL of medium containing 20 µg/mL F(ab')2-Goat anti-Human IgG, IgM (H+L) (Invitrogen) was added to each well. 72 hr later, cells were harvested at 500 g for 5 min, washed once with PBS, stained with 2 µg/mL CD69, CD86, CD81, or CD19 antibody in 20 mM HEPES buffer (pH 7.4), 150 mM NaCl, and 0.1% BSA for 20 min on ice, washed twice with PBS, and analyzed on a BD Accuri C6 flow cytometer.
3
Recombinant M25 Antibody Production
4
Transient Expression of Recombinant IgG
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For small scale transfections, 12 well plates of 293T cells at 80% confluence were transiently transfected with 0.5ug each of heavy and light chain vectors using polyethylenimine (PEI). After 16 hours, media was removed and replaced with serum-free media. After 3-4 days, supernatants were harvested and cell debris was removed by centrifugation at max speed in a microcentrifuge for 1 minute. For large scale transfections, expression vectors containing paired heavy and light chains were transiently transfected into 293T cells using polyethylenimine (PEI). Expression of recombinant full-length human IgG monoclonal antibodies were carried out in serum-free basal medium (Nutridoma-SP, Sigma-Aldrich). Four days after transfection, cell culture medium was collected and protein was purified using HiTrap™ Protein G HP column (1ml, GE Healthcare). Final IgG proteins were concentrated and buffer exchanged into 1x PBS using Millipore concentrator (30K MWCO). IgG protein concentration is determined by Nanodrop 2000 spectrophotometer.
5
Transient Transfection of 293T Cells for Recombinant IgG Production
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For small scale transfections, 12 well plates of 293T cells at 80% confluence were transiently transfected with 0.5ug each of heavy and light chain vectors using polyethylenimine (PEI). After 16 h, media was removed and replaced with serum-free media. After 3-4 days, supernatants were harvested and cell debris was removed by centrifugation at max speed in a microcentrifuge for 1 min. For large scale transfections, expression vectors containing paired heavy and light chains were transiently transfected into 293T cells using PEI. Expression of recombinant full-length human IgG monoclonal antibodies were carried out in serum-free basal medium (Nutridoma-SP, Sigma-Aldrich). Four days after transfection, cell culture medium was collected and protein was purified using HiTrap™ Protein G HP column (1ml, GE Healthcare). Final IgG proteins were concentrated and buffer exchanged into 1x PBS using Millipore concentrator (30K MWCO). IgG protein concentration is determined by Nanodrop 2000 spectrophotometer.
6
Monocyte-Endothelial Cell Co-Culture Assay
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To further investigate passage of lipoproteins through leaky endothelial cell junctions and their potential to transform macrophages into foam cells, co-cultures with human monocytes were performed. Transwell inserts with endothelial cells pre-incubated with everolimus (10 nM, 100 nM, 1 µM, 10 µM and 100 µM) for 24 hours were transferred to 12-well plates in which differentiated macrophages (see section “Cell culture eexperiments”) had been cultured for 5–8 days. Endothelial cells were carefully rinsed with PBS and supplied with fresh media before transferring them to monocyte cultures to avoid everolimus toxicity.
Prior to lipid loading, cell culture media was switched to serum free RPMI (including 1% Nutridoma-SP (Sigma-Aldrich, USA) and 1% penicillin-streptomycin). Endothelial cells were subsequently incubated with medium containing Alexa Fluor 488 AcLDL at 10 µg/ml concentration for 24 hours to allow trans-endothelial passage of AcLDL particles. Both supernatants (upper and lower chamber) were collected and the cells were fixed in 10% formalin for oil-red-O staining. A total of four independent experiments with different monocyte donors were performed.
Prior to lipid loading, cell culture media was switched to serum free RPMI (including 1% Nutridoma-SP (Sigma-Aldrich, USA) and 1% penicillin-streptomycin). Endothelial cells were subsequently incubated with medium containing Alexa Fluor 488 AcLDL at 10 µg/ml concentration for 24 hours to allow trans-endothelial passage of AcLDL particles. Both supernatants (upper and lower chamber) were collected and the cells were fixed in 10% formalin for oil-red-O staining. A total of four independent experiments with different monocyte donors were performed.
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