Inguinal and axillary lymph nodes from control or HFHF mice were collected in sterile PBS solution, and a single-cell suspension was prepared through a 100-μm cell strainer (Falcon, 352360). Cells were washed once in complete Dulbecco’s modified Eagle’s medium (DMEM) (Corning, 15-017-CV) containing glucose (4.5 g/liter), l -glutamine, sodium pyruvate, Hepes buffer, 10% fetal bovine serum, and antibiotic (streptomycin and penicillin) and then washed in PBS before CFSE labeling (BioLegend, 423801). CFSE-labeled cells were stimulated with PDIA3 peptide (0–2-20 μg/ml) in complete DMEM for 5 days and stained with 4′,6-diamidino-2-phenylindole (DAPI) (10 ng/ml; Sigma-Aldrich, D9542) alone or with DAPI plus CD4–allophycocyanin (APC)–Cy7 (BioLegend, 100526) and B220–Alexa Fluor 647 (BioLegend, 103226). Samples were acquired using Becton Dickinson Aria II (or Becton Dickinson Influx) and sorted as DAPI-negative (live) or CFSE-low (proliferated) CD4+ T cells or B220+ B cells.
B220 alexa fluor 647
Manufactured by BioLegend
B220–Alexa Fluor 647 is a fluorescently-labeled antibody that targets the B220 antigen. It is designed for use in flow cytometry applications to identify and enumerate B cells.
Automatically generated - may contain errors
Lab products found in correlation
7 aad, by Thermo Fisher Scientific (2 mentions)
100 μm cell strainer, by BD (1 mentions)
Aria 2, by BD (1 mentions)
Cfse labeling, by BioLegend (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Corning (1 mentions)
Rabbit anti c3, by Santa Cruz Biotechnology (1 mentions)
Streptavidin alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Thermo Fisher Scientific (1 mentions)
Indo 1 am, by Thermo Fisher Scientific (1 mentions)
Streptavidin pe cy7, by BioLegend (1 mentions)
Cd3 apc cy7, by BioLegend (1 mentions)
Cd3 b220, by BD (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Alexafluor 700 cd3, by BioLegend (1 mentions)
Cd19 pe, by BioLegend (1 mentions)
Efluor 780, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa x 20 flow cytometer, by BD (1 mentions)
4 protocols using b220 alexa fluor 647
1
Lymph Node Cell Isolation and Proliferation
2
Immunofluorescence Analysis of Spleen and Kidney
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The spleens and kidneys from 52-wk-old control (Ptpn1f/f and mb1cre) and Ptpn1f/f-mb1cre mice were frozen in Cryoblock (Medite). (Spleens were fixed before with 4% paraformaldehyde and incubated overnight at 4°C in 30% sucrose in PBS.) 8-µm sections were placed on glass slides, and spleen samples were stained with PNA-biotin followed by streptavidin–Alexa Fluor 555 (Invitrogen) and B220–Alexa Fluor 647 (BioLegend), whereas kidney sections were stained with anti–mouse IgG-Cy3 (Invitrogen), rabbit anti-C3 (Santa Cruz Biotechnology, Inc.), and donkey anti–rabbit Alexa Fluor 488 (BioLegend). Nuclei were stained with DAPI, and the slides were analyzed on an LSM780 confocal microscope (objective: 20×/0.8). Images were acquired by the ZEN 2010 software.
3
Measuring B Cell Calcium Flux
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Mouse peripheral blood was processed to remove RBC via lysis prior to Fc blocking and then stained with antibodies against surface markers including biotin-CD93 (Invitrogen, cat # 13-5892-85), BV605-CD19 (BD, cat # 115539), APC Cy7-CD3 (BioLegend, cat # 100222), Alexa Fluor 647-B220 (BioLegend, cat # 103226), and PE Cy7-streptavidin (BioLegend, cat # 405206). The cells were washed with complete RPMI-1640 pre-warmed to 37°C and stained with Indo-1 AM (Invitrogen, cat # I1223) diluted to 2 μM in warm RPMI-1640 and incubated at 37°C for 30 min before washed and stained with 7-AAD (Invitrogen, cat # A1310).
For acquisition, data scales of the detectors for the BUV395 (379/28) and BUV496 (515/30 450LP) filters were set to linear. Samples were kept at 37°C prior to and during acquisition. Baseline was recorded for 30 sec, followed by the addition of anti-mouse IgM (Jackson ImmunoResearch; cat # 115-006-075) to a final concentration of 10 μg/mL, immediately mixed before resuming acquisition for further 3.5 mins to measure the calcium flux following BCR ligation. At 4 mins post-acquisition, ionomycin (Invitrogen, cat # I24222) was added to the sample tube to a final concentration of 1 μg/mL, immediately mixed before before resuming acquisition for another 4 mins to measure maximal Ca2+ flux output.
For acquisition, data scales of the detectors for the BUV395 (379/28) and BUV496 (515/30 450LP) filters were set to linear. Samples were kept at 37°C prior to and during acquisition. Baseline was recorded for 30 sec, followed by the addition of anti-mouse IgM (Jackson ImmunoResearch; cat # 115-006-075) to a final concentration of 10 μg/mL, immediately mixed before resuming acquisition for further 3.5 mins to measure the calcium flux following BCR ligation. At 4 mins post-acquisition, ionomycin (Invitrogen, cat # I24222) was added to the sample tube to a final concentration of 1 μg/mL, immediately mixed before before resuming acquisition for another 4 mins to measure maximal Ca2+ flux output.
4
Murine Splenic B Cell Sorting and Apoptosis
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Spleens were collected from Sh2b3+/+ and Sh2b3E372K/E372K mice and meshed to generate single-cell suspension, which were stained with antibody cocktail containing BV480-CD93 (BioLegend; cat # 136507), PE-CD19 (BioLegend; cat # 115508), Alexa Fluor 647-B220 (BioLegend; cat # 103226) and Alexa Fluor 700-CD3 (BioLegend; cat # 100216) antibodies, then with 7-AAD (Invitrogen; cat # A1310). The cells were sorted into live (7-AAD-) transitional (CD19+CD3-B220+CD93+) and mature B cells (CD19+CD3-B220+CD93-) on BD FACSAria Fusion and FACSAria II (BD), before cultured in complete RPMI-1640 media without/with 5 μg/mL goat anti-mouse IgM (Jackson ImmunoResearch; cat # 115-006-075) and/or recombinant murine 25 ng/ml IL-4 (Peprotech; cat # 214-14) for 16 hours at 37°C/5% CO2 before staining with fixable viability dye eFluor 780 (Invitrogen; cat # 65-0865-14) and FITC-Annexin V (BD; cat # 556419) for apoptosis analysis on a LSRFortessa X-20 flow cytometer (BD). To determine BAFF-R experssion, sorting and cell culture was performed in as described above for the apoptosis assay. Following 16-hr incubation at 37°C/5% CO2, the cultured B cells were stained with Fc block followed by fixable viability dye eFluor 780 and FITC BAFF-R monoclonal antibody (Invitrogen; cat #11-5943-81) and analyzed on a LSRFortessa X-20 flow cytometer (BD).
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