Peripheral blood mononuclear cells (PBMCs) were isolated from normal healthy donor leukopacks (Children’s Hospital Blood Donor Center, Boston, MA; OneBlood, Inc., Miami, FL) and tonsillar mononuclear cells were isolated from discarded tonsil specimens provided by Nicklaus Children’s Hospital (Miami, FL) using Histopaque-1077 (Sigma-Aldrich, Milwaukee, WI) gradient centrifugation and then stored in liquid nitrogen for future use in flow cytometry and in B cell isolation, adhesion and transcriptional analyses (1 (link), 54 (link)). To isolate resting naïve B cells, PBMC aliquots were thawed, washed and subjected to a combination of immunomagnetic bead cell separation kits: Human Naïve B cell Isolation Kit II and CD27 MicroBeads Kit (Miltenyi Biotec, Auburn, CA). To validate B cell and naïve B cell isolation, PBMCs and resting naïve B cell isolates were stained with a combination of anti-CD3-APCCy7, anti-CD19-PerCP, anti-CD27-PECy7, anti-IgD-APC and Zombie Green viability dye (Biolegend, San Diego, CA) and analyzed by flow cytometry, confirming purity of IgD+/CD19+/CD27− naïve B cells of >95%.
Human na ve b cell isolation kit 2
Manufactured by Miltenyi Biotec
Sourced in Germany
The Human naïve B cell isolation kit II is a laboratory tool designed for the isolation of human naïve B cells from peripheral blood mononuclear cells (PBMCs). The kit utilizes a combination of magnetic bead-based cell separation techniques to selectively enrich for naïve B cells, which can be further used for various downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd19 percp, by BioLegend (1 mentions)
Zombie green viability dye, by BioLegend (1 mentions)
Anti cd3 apc cy7, by BioLegend (1 mentions)
Anti cd27 pe cy7, by BioLegend (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Cd40l, by Thermo Fisher Scientific (1 mentions)
Il 21, by Miltenyi Biotec (1 mentions)
Lymphocyte separation medium, by Corning (1 mentions)
Rosettesep, by STEMCELL (1 mentions)
Na ve cd4 t cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Cpg odn 2006, by InvivoGen (1 mentions)
Aim 5 medium, by Thermo Fisher Scientific (1 mentions)
Tgf β, by R&D Systems (1 mentions)
Il 12, by R&D Systems (1 mentions)
4 protocols using human na ve b cell isolation kit 2
1
Isolation and Characterization of Naïve B Cells
2
Activation of Naïve B Cells into Plasmablasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For 24 or 48 h cultures, PBMC was thawed and stimulated with IL-21 (50 ng/mL) (Miltenyi) with and without CD40L (5 μg/mL) (Peprotech). Four hours prior to harvest, brefeldinA/monensin were added to the cultures. Cells were harvested and stained for flow cytometry.
For plasmablast cultures, naïve B cells were isolated using the human naïve B cell isolation kit II (Miltenyi) and purity was assessed following 12-day cultures. Samples with purity >90% were plated at two million cells per well and cultured with IL-21 (15 pg/mL or 50 ng/mL) (Miltenyi) and CD40L (5 μg/mL) (Peprotech).
For plasmablast cultures, naïve B cells were isolated using the human naïve B cell isolation kit II (Miltenyi) and purity was assessed following 12-day cultures. Samples with purity >90% were plated at two million cells per well and cultured with IL-21 (15 pg/mL or 50 ng/mL) (Miltenyi) and CD40L (5 μg/mL) (Peprotech).
3
Isolation of Immune Cell Subsets
4
Induction of Autoantigen-Specific Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The DCs were induced as above and stimulated with different NitraTh-based vaccines (HER2, HER2-Th, HER2-NitraTh, CB1, CB1-Th, or CB1-NitraTh). Naïve CD4+ T cells sorted from the same donor were cocultured with these DCs in the AIM-V medium (Gibco, USA) containing 0.5 ng/mL IL-12 and 1 ng/mL TGF-β (R&D Systems, USA) for 7 days. Culture medium and cytokines were renewed every 3 days. On day 5, human naïve B cells (purity of >95%, viability of >90%) that were sorted by Human Naïve B Cell Isolation Kit II (Miltenyi, Germany) were activated with CpG ODN 2006 (InvivoGen, USA) and NitraTh-based vaccines mentioned above. On day 7, B cells were harvested and cocultured with DC-T cells. At 12 days later, the culture supernatants and cells were collected, and autoantigen-specific antibodies and antibody-secreting B cells were measured by ELISA and ELISpot assay, respectively.
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!