All mouse experiments were approved by the appropriate institutional and governmental review committees for animal welfare (Thüringer Landesamt für Verbraucherschutz; breeding license 02-052/16). All mouse lines were on a C57BL7/6 background. Primary B cells were isolated from spleens of 8- to 16-week-old mice. Briefly, single cell suspensions were prepared from spleens, depleted of red blood cells using Red Blood Cell Lysis Buffer (Sigma) and purified by negative selection using MACS with anti-CD43 beads (Miltenyi Biotec) according to the manufacturer’s instructions. Isolated B cells were then cultured at 5x105 cells/mL in 24-well plates for 5 days at 37°C in RPMI (Invitrogen) supplemented with 10mM HEPES (ThermoFisher), 50mM β-mercaptoethanol (Sigma), 100U/mL Pen Strep (Gibco) and 10% FBS (Sigma) (cRPMI). Stimulating conditions were achieved by adding 10µg/mL LPS (Sigma, L4391) or 1µg/mL anti-CD40 (eBioscience) with 20ng/mL IL-4 (eBioscience). 3 to 4 hours after seeding, 30µg/mL Zymosan (Wako) or 500µg/mL heat-killed Candida albicans yeast or hyphae were added to the culture. Cultured cells were fed at day 3, maintaining the same culture conditions. After 3 and 5 days of culture, cells were harvested and supernatants stored at -20°C and -80°C for further analysis.
Anti cd43 beads
Manufactured by Miltenyi Biotec
Anti-CD43 beads are a type of magnetic bead that can be used to isolate and enrich CD43-positive cells from a heterogeneous cell population. The beads are conjugated with an antibody that specifically binds to the CD43 antigen expressed on the cell surface.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharides (lps), by Merck Group (7 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (7 mentions)
Hepes, by Thermo Fisher Scientific (6 mentions)
2 β mercaptoethanol, by Thermo Fisher Scientific (4 mentions)
Cd4 t cell isolation kit, by Miltenyi Biotec (3 mentions)
Anti cd40, by Thermo Fisher Scientific (2 mentions)
Anti cd11c beads, by Miltenyi Biotec (2 mentions)
Np 25 klh, by Biosearch Technologies (2 mentions)
Imject alum, by Thermo Fisher Scientific (2 mentions)
Anti pe magnetic beads, by Miltenyi Biotec (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Red blood cell lysis buffer, by Merck Group (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Zymosan, by Fujifilm (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Ld column, by Miltenyi Biotec (1 mentions)
Soluble anti cd28, by BioXCell (1 mentions)
Anti biotin bead, by Miltenyi Biotec (1 mentions)
21 protocols using anti cd43 beads
1
Purification and Stimulation of Primary Mouse B Cells
2
Isolation and Stimulation of Murine Naïve B Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Naïve resting B cells from splenocytes of 8-week-old male C57BL6 mice were isolated with anti-CD43 beads (Miltenyi), confirmed as 95 % CD19+ by flow cytometry (FACS Calibur), and resuspended in cold media with either 10 ug/ml anti-mouse IgM goat IgG Fab fragments (Jackson Immunology) or 25 ug/ml Salmonella typhimurium typhus LPS (Sigma) were added. The cells were rested on ice for 30 min following a previously published method [72 (link)] and incubated at 37 °C/5 %CO2 for the experimental times. Animal care and use in this study are covered under the “Assurance of Compliance with PHS (USA) Policy on Humane Care and Use of Laboratory animals by Awardee Institutions” and approved by the Institutional Animal Care and Use Committee of Tufts University (Animal Welfare Assurance Number A-3775-01).
3
Isolation of Purified B Cells from Splenocytes
4
Primary B Cell Activation Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For LPS/IL4 cultures, primary B cells were purified by immunomagnetic depletion using anti-CD43 beads (Miltenyi Biotec) and cultured at a final concentration of 1.2 × 106 cells per ml in complete RPMI supplemented with 10% FBS, 50 μM of 2-βMercaptoethanol (Gibco), 20 mM Hepes (Gibco), 10 ng ml−1 of IL4 (PeproTech) and 25 μg ml−1 lipopolysaccharide (LPS, Sigma-Aldrich). In the T–B-cell co-culture, CD43+ cells were added at a ratio 1:1 to CD43− B cells in the presence of plastic-bound anti-CD3 (Tonbo, 5 μg ml−1) and soluble anti-CD28 (BioXcell, 2 μg ml−1) for T-cell stimulation. When indicated, cells were treated with anti-mouse CD40 (BD Pharmigen, 1 μg ml−1).
5
Isolation of Immune Cell Subsets from Murine Spleen
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To isolate DCs, spleens were collected, incubated for 30 min at 37 °C in HBSS (Gibco) supplemented with CaCl2, MgCl2, and collagenase D at 400 U ml−1 (Roche). After digestion, tissue was forced 5 times through a 21-gauge needle and filtered through a 70 μm strainer into a 15 ml falcon tube with PBS supplemented with 0.5% BSA and 2 mM EDTA (PBE). Red-blood cells were lysed with ACK buffer (Gibco), and the resulting cell suspensions were filtered through a 70-μm mesh into PBE. DCs were obtained by magnetic cell separation (MACS) using anti-CD11c beads (Miltenyi Biotec), as per the manufacturer’s instructions. To isolate CD4+ T cells, CD8+ T cells, and B cells, spleens were processed as above, without collagenase digestion. CD4+ T cells and CD8+ T cells were isolated by negative selection using a cocktail of biotinylated antibodies targeting Ter119, CD11c, CD11b, CD25, B220, NK1.1, and either CD8 (for CD4+ isolation) or CD4 (for CD8+ isolation), followed by anti-biotin beads (Miltenyi Biotec), as per the manufacturer’s instructions. B cells were obtained by negative selection using anti-CD43 beads (Miltenyi Biotec), as per the manufacturer’s instructions.
6
Isolation and Stimulation of Mouse B Cells
7
Generating NP-specific Plasma Cells
8
Generating NP-specific Plasma Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To generate NP-specific PCs of different ages, B1-8hi B cells were isolated from spleen and lymph nodes by magnetic cell separation (MACS) using anti-CD43 beads (Miltenyi Biotec), and transferred intravenously to C57BL/6 recipient mice with 3-7x106 B1-8hi B cells or 0.5-1x106 Igλ+ B1-8hi B cells per mouse. One day after the transfer, recipient mice were immunized intraperitoneally with 50 μg of NP(25)-KLH (Biosearch Technologies) emulsified in alum (Imject Alum; Thermo Fisher Scientific) at 2:1 v:v ratio in 150 μL volume for 2-8 weeks.
9
Sequencing Sμ Mutations in Activated B Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Analysis of mutations at Sμ by NGS was performed as described in Pérez-Durán P et al.51 (link). Briefly, naive B cells were purified by immunomagnetic depletion with anti-CD43 beads (Miltenyi) and cultured with 25 μg/ml LPS (Sigma) plus 10 ng/ml IL4 (PeproTech) for 3 days to obtain activated B cells. Immature B-cell populations were purified by FACs-sorting as follows: B220+ CD19−, pre-pro-B; B220+ CD19+ IgM-CD25− pro-B; B220+ CD19+ IgM-CD25+ pre-B; B220+ CD19+ IgM+ IgD−, immature. Genomic DNA from purified B-cell populations was extracted, and PCR amplified with Pfu Ultra high-fidelity DNA polymerase (Stratagene). PCR reactions were fragmented with Covaris, and adapter-ligated libraries were generated, processed, and sequenced on a HiSeq2500 according to the manufacturer’s instructions (Illumina).
10
Cell Culture and B Cell Stimulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
3T3-L1 fibroblast cells were a gift from the Life Science Experimental Teaching Demonstration Center of Southern University of Science and Technology, and were cultured with DMEM high glucose liquid media (Hyclone) containing 10% fetal bovine serum (ExCell Bio), 100 U/ml penicillin and 100 μg/ml streptomycin (Hyclone) in 24-well plates or 100 mm dishes (NEST Biotechnology) at 37°C with 5% (v/v) CO2. HEK 293 T cells were cultured under the same conditions as 3T3-L1 fibroblast cells.
For B cell stimulation in vitro, anti-CD43 beads (Miltenyi Biotec) and LS columns (Miltenyi Biotec) were used for the purification of splenic B cells. Two million purified splenic B cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin and 100 μg/ml streptomycin (Hyclone). LPS (20 µg/ml, Sigma) was used to stimulate B cells for retroviral transduction. Anti-CD40 (1 µg/ml, BD Biosciences) antibody was used for immunoblot analysis of NF-κB pathway in splenic B cells upon CD40 engagement.
For B cell stimulation in vitro, anti-CD43 beads (Miltenyi Biotec) and LS columns (Miltenyi Biotec) were used for the purification of splenic B cells. Two million purified splenic B cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin and 100 μg/ml streptomycin (Hyclone). LPS (20 µg/ml, Sigma) was used to stimulate B cells for retroviral transduction. Anti-CD40 (1 µg/ml, BD Biosciences) antibody was used for immunoblot analysis of NF-κB pathway in splenic B cells upon CD40 engagement.
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!