Splenic B cells were purified by negative selection (eBioscience Magnisort Mouse B cell enrichment kit). B cell purity measured by FACS analysis (FACSCalibur and CellQuest software; BD Biosciences) using anti-B220 antibody (Biolegend) yielded >95% B220+. Purified B cells were seeded at a final concentration of 1.5 × 10^6 cells per milliliter. For class switching experiments, B cells were stimulated 5 μg/mL anti-CD40 (HM40–3) agonistic antibody (eBioscience), or 5 μg/mL LPS (Sigma), together with 5 ng/mL mIL-4 (R&D Systems) for 96 h to induce switching to IgG1. 5 μg/mL LPS (Sigma) induced switching to IgG3. 5 μg/mL LPS (Sigma) with 5 ng/mL mIL-4 (R&D), 5 ng/mL mIL-5 (Tonbo Bioscience), 5 nM retinoic acid (Sigma Aldrich) and 5 ng/mL TGF-β (Thermo Fisher Scientific) induced switching to IgA. All cells were cultured with RPMI 1640 supplemented with 10% (vol/vol) heat-inactivated FCS, 5 mM Hepes, 2 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin, 50 μM 2-mercaptoethanol.
Mil 4
Manufactured by R&D Systems
The MIL-4 is a compact, automated laboratory instrument designed for cell culture and biological analysis. It features precise temperature and environmental control, enabling consistent and reproducible experimental conditions. The MIL-4 is intended to facilitate various cell-based assays and applications in the life sciences research and clinical laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (3 mentions)
Lipopolysaccharides (lps), by Merck Group (3 mentions)
Magnisort mouse b cell enrichment kit, by Thermo Fisher Scientific (2 mentions)
Anti b220 antibody, by BioLegend (2 mentions)
Anti cd40 hm40 3 agonistic antibody, by Thermo Fisher Scientific (2 mentions)
Cellquest, by BD (2 mentions)
M csf, by R&D Systems (2 mentions)
Interleukin 4 (il 4), by R&D Systems (2 mentions)
Anti cd40 antibody, by Thermo Fisher Scientific (1 mentions)
Anti iga pe, by Southern Biotech (1 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (1 mentions)
Tgf β, by R&D Systems (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Mouse granulocyte macrophage colony stimulating factor mgm csf, by R&D Systems (1 mentions)
Z 4 hydroxytamoxifen 4 oht, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Cd19 isolation kit, by Miltenyi Biotec (1 mentions)
Recombinant mouse il 2, by R&D Systems (1 mentions)
9 protocols using mil 4
1
Induction of Immunoglobulin Class Switching in Murine B Cells
2
Purification and Stimulation of Murine Splenic B Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse splenic B cells were purified by negative selection (eBioscience Magnisort Mouse B cell enrichment kit). B-cell purity was >95% as measured by FACS analysis (FACSCalibur and CellQuest software; BD Biosciences) using anti-B220 antibody (BioLegend). Purified B cells were seeded at a final concentration of 0.5 or 0.25 × 106 cells/mL. For plasmablast differentiation, B cells were stimulated with 5 µg/mL LPS (Sigma) for 72 h, and for IgG1 CSR, B cells were stimulated 5 µg/mL anti-CD40 (HM40-3) agonistic antibody (eBioscience), or 5 µg/mL LPS (Sigma), together with 5 ng/mL mIL-4 (R&D Systems) for 96 h. All B cells were cultured in RPMI 1640 supplemented with 10% (vol/vol) heat-inactivated FCS, 5 mM Hepes, 2 mM l -glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 50 µM 2-mercaptoethanol.
3
Measurement of Class Switch Recombination
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Class switch recombination assays were performed essentially as previously described (Noordermeer et al, 2018 (link)). 1 × 105 CH12F3‐2 cells were plated in 24‐well plates in growth medium supplemented with 1 μg/ml anti‐CD40 antibody (eBioscience), 1 ng/ml TGF‐β (R&D Systems), and 10 ng/ml mIL‐4 (R&D Systems). After 48 h, cells were harvested, stained with anti‐IgA‐PE (Southern Biotech), and fixed with 4% formaldehyde. Fluorescence signal was acquired on an Attune NxT Flow Cytometer (Thermo‐Fisher). Data were analyzed using FlowJo software (BD Biosciences).
4
Bone Marrow-Derived Macrophage Differentiation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Analysis of bone marrow-derived macrophages was performed as previously described18 (link). Bone marrow cells were isolated from 8-week-old female C57BL/ 6 J mice. They were plated at 2 × 106 cells per 6 cm dish and differentiated into macrophages in DMEM supplemented with 20 ng/mL macrophage colony-stimulating factor (M-CSF) (R&D Systems, Minneapolis, MN, USA) at 37 °C in 5% CO2 for seven days. The macrophages were incubated with serum-free DMEM (M0), serum-free DMEM with 20 ng/mL IL-4 (R&D Systems, M(IL-4)), or SHED-CM(M(SHED-CM)) for 24 h, followed by mRNA analysis.
5
Generation and Purification of Flt3L-DCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BMDCs were cultured as previously described with minor modification [59 (link)]. Bone marrow cells collected from femurs and tibias were cultured in RPMI-1640 medium (HyClone) supplemented with 10% FBS (Gibco), 50 μM 2-mercaptoethanol (Sigma–Aldrich), penicillin and streptomycin (Invitrogen) in the presence of 10 ng/ml mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF; R&D) and 10 ng/ml mIL-4 (R&D). The culture medium was replenished on Day 3, and the cells were harvested on Day 7. Bone marrow cells were cultured in the presence of 150 ng/ml mFlt3L (R&D) and harvested on Days 8−9 to collect Flt3L-DCs. For p38α deletion in Flt3L-DCs derived from p38αCreER mice, 0.5 μM (Z)-4-hydroxytamoxifen (4-OHT; Sigma–Aldrich) was added on Day 4. The purity of both CD11c+ BMDC populations was >80%. Flt3L-cDC1s (CD11c+ B220−CD24+CD11b−) were sorted by FACS for coculture.
6
Th2 Differentiation of Naive CD4+ T Cells
7
Multiparameter Flow Cytometry Phenotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
mAbs specific for mouse B220 (RA3-6B2), c-Kit (2B8), CD3ε (145-2C11), CD5 (53-7.3), CD8 (53-6.7), CD11b (M1/70), CD11c (HL3), CD16/CD32 (2.4G2), CD19 (1D3), CD25 (PC61), CD34 (RAM34), CD45.1 (A20), CD45.2 (104), CD49b (DX5), CD127 (A7R34), GATA3 (L50-823), Gr-1 (RB6-8C5), MHC class II (M5/114.1), NK1.1 (PK136), Sca-1 (D7), Siglec-F (E50-2440), ST2 (U29-93), Thy1.2 (53-2.1), IL-4 (11B11), IL-12 (C15.6), IFN-γ (XMG1.2), and fluorochrome-conjugated streptavidin were purchased from BD Biosciences. mAbs specific for mouse CD4 (GK1.5), Flt3 (A2F10), F4/80 (BM8), IL-5 (TRFK5), IL-17RB (9B10), and NKp46 (29A1.4) were purchased from BioLegend. mAbs specific for mouse α4β7 (DATK32), FcεRIα (MAR-1), IL-13 (eBio13A), and killer cell lectin-like receptor G1 (KLRG1; 2F1) were purchased from eBioscience. mAbs against mouse CD16/CD32 (2.4G2), CD28 (37.51), and erythroid cell marker (TER-119) were purified from hybridoma culture supernatants in our laboratory.
Recombinant mouse IL-2, mIL-4, mIL-6, mIL-7, mIL-33, and mTGF-β1 were purchased from R&D Systems. p38 inhibitor (SB203580) and actinomycin D were purchased from Sigma-Aldrich.
Recombinant mouse IL-2, mIL-4, mIL-6, mIL-7, mIL-33, and mTGF-β1 were purchased from R&D Systems. p38 inhibitor (SB203580) and actinomycin D were purchased from Sigma-Aldrich.
8
Isolation and Culture of Murine B Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Equal numbers of male and female B6 mice were used for isolation of B cells. Splenic B cells were purified using The EasySep™ Mouse B Cell Isolation Kit (Stemcell Technologies Kent, WA). B-cell purity measured by FACS analysis (FACSCalibur and CellQuest software; BD Biosciences) using anti-B220 antibody (eBioscience, ThermoFisher Scientific, Waltham, MA) yielded >95% B220+. Purified B cells were seeded at a final concentration of 1.5 x 106 cells per milliliter. For class switching experiments, B cells were stimulated 5 μg/mL anti-CD40 (eBioscience) agonistic antibody, or 5 μg/mL LPS (MilliporeSigma), together with 5 ng/mL mIL-4 (R&D Systems) for 96 h to induce switching to IgG1. All cells were cultured with RPMI 1640 supplemented with 10% heat-inactivated FCS, 5 mM Hepes, 2 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin, 50 μM 2-mercaptoethanol. To evaluate B-cell survival ex vivo, purified B cells from mice of different genotypes were treated with 4OHT in vitro in the presence of BAFF (100ng/ml, R&D Systems). For proliferation assays, B cells were stimulated with anti-IgM (10μg/ml, Jackson ImmunoResearch Laboratories, West Grove, PA) and IL4 (10ng/ml, R&D Systems). For pre-B cell culture, bone marrow cells were cultured for 5 days in the absence or presence of recombinant murine IL-7 (10ng/ml).
9
Macrophage Polarization Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The spleen cells were washed three times and cultured in a 12-well tissue culture in RPMI-1640 supplemented with 10% FBS and antibiotics (penicillin/streptomycin, Invitrogen) and 100 ng/ml of M-CSF (R&D Systems, France) at 37°C in a humidified atmosphere containing 5% CO2 for 7 days to induce full macrophage differentiation and maturation. Macrophage polarisation was obtained by removing the culture medium and culturing cells for an additional 24 h in RPMI-1640 supplemented with 10% FBS and antibiotics and 100 ng/ml LPS (derived from E. coli 0111:B4, Invivogen) plus 20 ng/ml IFN-γ (for M1 polarisation, M(LPS-IFN-γ)) or 20 ng/ml IL-4 (for M2 polarisation, M(IL-4)) (R&D Systems). The cells were harvested for RT-qPCR analysis.
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!