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B Cells - Science topic

B cells: their development, function and signal transduction
Questions related to B Cells
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Hi all,
I am planning on NP-imunisation of mice to study B cells. However, when looking at suppliers, there are various ratio's available. Which one is recommended, or what is the rationale to order one or the other? E.g. NP:CGG 7, or 26, or even >40?
Best,
Chris
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Unfortunately, there is no straightforward answer to your question, as it largely depends on the mouse strains used for your experiments and your final readout for the characterization of the B cell response.
If you fancy memory B cells a NP:CGG ratio > 26 is a good starting point am Mark Shlomchik's work is a good starting point. Please see the attached paper for more details and future reference.
For a quick characterization of the B cell response/ B cell compartment check out:
I hope that helps!
All the best & good luck with your experiments,
Michael
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The human B-cell antibody CD79a doesn't work
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You might want to look at Washington State University's Monoclonal Antibody Center, as they have a good collection of mAbs vs many domestic species antigens, including bovine:
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B cells
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This seems like an exam or homework question for a class.
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Dear colleagues,
I am writing a paper on non-specific antibody binding to self-antigens. Do you know whether maturing B-cells are exposed to all self-antigen peptides possible (trillions or quadrillions of peptides) or only to a fraction of peptides that are most common on the cell surface/inside cells that are "exposed" for binding of the antibody? For example, would B-cells be exposed to histone peptides that are exposed for antibody binding in natural setting (non-cryptic epitopes), whereas cryptic epitope peptides of the same histone would not be shown to B-cells?
Thanks,
Kind regards,
Maria
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Anding Shen 's answer is a bit too simplistic. New B cells are generated constantly and pass through a 'window' of tolerance induction as they are acquiring surface antibody. If they encounter an antigen they bind with sufficient affinity to activate the B cell, the B cell will endocytose it, process it and present antigenic peptides on its surface, looking for T cells to recognize the antigen. If there is recognition by T cells, the B cells is driven differentiate and produce antibody. If no help is provide by T cells, the activated nascent B cell dies.
Research has shown that many autoantigens are not tolerized out - the B cell never encounters them during maturation. Once mature the primary B cells cannot be tolerized. Secondary B cells undergo a second tolerance window after primary activation and can be tolerized if the activated B cell does not receive help from T cells.
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I transduced my B cells with GFP containing plasmids using lentivirus and achieved 60% positivity. However, when I checked my cells under the confocal microscope, I can not see any GFP signals. (Maybe a little bit, but the signal it gave is similar to the signal given by non-transduced B cells). I was wondering does anyone know what could happened?
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Flow cytometer is much more sensitive than confocal microscope. I have several experiemce when a weak GFP-expressing cell is easily detectable by flow cytometer but direct imaging cannot see anything. Try increase your microscope laser intensity may help a little, but background will increase too.
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I'd like to know the Highest protein producing cell in the Body ?
I guess It's B cells which produce antibodies, Any other comments ?
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Hepatocytes in the liver express and secrete a great deal of protein, including serum albumin, which is the most abundant protein in blood plasma.
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I have some flow cytometry issues with T-bet staining, thus I would like to ask about the transcription factor's approximate percentage (a mean, actually ) in peripheral blood samples from healthy individuals...
Thank you in advance
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Fabian Flores-Borja Thank you for the reply!
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Is it logical that I isolate sp B cells from f/f Aicda-cre and in vitro add cytokines to stimulate to undergo CSR? Or this cre only works for in vivo immunization?
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Hi, I have been struggling with getting a decent staining of CD19 on my PBMCs derives human B cells. I have tried Spark NIR CD19 from biolegend (cat 302269) and Alexa Fluor 700 CD19 also from biolegend (cat 302225) and couldn’t get a decent stain from the first one and had to use the second one in 1:10 dilution. I am using a 3 Lasers (red, blue, violet) northern lights 3000 flow cytometer from Cytek.
does anyone have any recommendations on another antibody or on how to improve my staining? I was looking into trying CD20 instead but from what I understood it’s not a pan B Cell marker
thanks!
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"Pan-B cell" markers are all deficient in at least one part of B cell development whether pre- pre-pro pro- or plasma. CD19 should capture what I think you are looking for, but so will CD20, and also CD79b, which is part of the BCR signaling complex.
Monoclonal Mouse IgG1 Clone # 4G7-2E3 works very well in our hands if you'd just like a clone (CD19) and 2H7 is good for CD20 with the added note that it also works with NHP samples.
As far as labeling, I'd need a bit more detail in terms of what you're doing. We do quite a bit of flow and have several cyteks, although the particular cytometer should not make much of a difference...
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Activation of naive T cells generally requires antigen to be presented by dendritic cells in lymph nodes. Activation of naive B cells generally requires opsonized antigen to be displayed by follicular dendritic cells in lymph nodes.
Thus, it seems that dendritic cells and complement (innate immune system) need to recognize a pathogen before an adaptive immune response can be initiated. Is this always the case?
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B1 b cells can respond to pathogens present in body cavity fluids indepenantt of t cells or innate immune cells. They make low affinity igm and have no memory. So yes.
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Dear colleagues,
I'm looking for assays to measure the effects of treatments on IgE release by B cells. Isolation of primary B cells from mouse spleen or human blood, then cultivation, stimulation protocols. Does anyone know whether Interleukin-4 is sufficient (it is known to stimulate IgE release)?
Thank you very much,
Best wishes,
Tineke Vogelaar
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I briefly wanted to reach out concerning your inquiry. I would suggest to use the protocol the protocol from Gallagher et al. and combine IL-4 with anti-CD40 for stimulation.
As a negative control you may want to consider titrating in IL-21 as this will potently suppress IgE production as per
which broadly uses a similar B cell culture protocol. For a nice, fast and easy CSR flow cytometry staining protocol (albeit not specifically for IgE) check:
I hope that helps and good luck for your experiments.
All the best & kind regards,
Michael
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I noticed that researchers use MOG35-55 to induce a type of T cell-dependent EAE, while for both T cell and B cell-dependent EAE, they choose rhMOG. Some papers report that B cells also contribute to the disease process in rmMOG-induced EAE setting. If these two recombinant proteins both activate B cells, I want to know what's the difference between them.
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Dear Echo Zhu
Please see the comparison below. There is a single amino acid substitution at position 42 of the MOG protein (position 7 of the peptide respectively) between the rodent (mouse/ rat) and human MOG protein from serine to proline.
rmMOG vs. rhMOG (aa 35-55)
rmMOG Met-Glu-Val-Gly-Trp-Tyr-Arg-Ser-Pro-Phe-Ser-Arg-Val-Val-His-Leu-Tyr-Arg-Asn-Gly-Lys
rhMOG Met-Glu-Val-Gly-Trp-Tyr-Arg-Pro-Pro-Phe-Ser-Arg-Val-Val-His-Leu-Tyr-Arg-Asn-Gly-Lys
The difference in B cell dependency of MOG induced EAE seems to majrily be attributed to immunization with full length (human) protein vs. the rodent peptide (in C57BL/6 mice), which may differ in other mouse strains (given their different MHCII) as per:
I hope that helps.
All the best and good luck with your experiments,
Michael
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Hello everybody,
I am doing immunocytochemistry staining for splenic B cells from WT and Knockout mice for one protein. By FACS analysis, it's clear that the signal for the knockout protein is nearly absent in Knockout cells, but when I do confocal microscopy experiment for the same cells, the signal seems to be the same in knockout as in WT. Does anyone have an explanation or can recommend what to do?
P.S: I use the same primary antibody for IF and ICC and No background for the secondary antibody in the control.
Thank you!
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Thank you Alibek, I will do it and I hope it will work :)
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What would be ideal mice strain to study T-cell dependent B-cell activation or KLH immunization?
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You may use C57BL/6 mice.
Please refer to the two articles attached below.
Best.
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In human PBMC cell cultures, is it necessary to use anti-Ig (in conjunction with other stimuli, such as IFNγ, IL-21and/or R848) to induce CD11c+T-bet+ B cells?? The cytokines and the TLR7-ligand alone , are not capable of inducing these cells??
Thank you in advance!!
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It really depends on the B cell population you begin the culture with. My cultures were sort purified prior to culturing. I found that BCR engagement was not absolutely necessary for cultures generated from memory subsets. Cultures that were initiated from naive B cells needed the BCR stimulation. However, the TLR7 and IL-21 signaling were essential for the development of the CD11c+ phenotype within the system.
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We are generating bone marrow chimeric mice using the X-ray irradiation. It worked fine when GammaCell 40 was used. However, our institute recently replaced that machine with a new machine "X-RAD 225 XL", somehow our chimeras don't show a B cell population after 6 weeks, at which time we usually check our chimeras and they are well reconstituted. We are thinking the problem may be the new machine is not working in this particular case. Could you guys help me with this? Thanks.
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I think, u can check out the irradiation dose released by the machine, using dosimetric film?
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I want to simulate a germinal center reaction with human cells in vitro. Ideally, I want to use human naive B cells, and simulate Antigen-stimulation and further GC reaction in vitro in order to look at AG-specific clonal B cell expansion, SHM and CSR.
From what I read CD40L supports BC growth in vitro nicely, but how can I induce AG-specific clonal expansion from a relatively small sample of PBMC (say 30 ml of whole blood, or 10 million PBMC)? I want to generate clonal expansion, and I think CPG works for polyclonal expansion, correct? Can you recommend a model antigen to add to PBMC to mimic GC reaction, for example Influenza or CMV particle? And will these stimulate naive B cells? Are the reagents commercially available? Also is there a commercially available CD40L-expressing human fibroblast feeder cell line?
Many, many thanks
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A couple of years later... I am facing the same doubts! It would be great if Louisa or Alexander could send me those papers!! ;)
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This one is for the B cell immunologists. In the absence of an animal model or antigen-experienced samples, is it possible to robustly induce antigen-specific antibodies from naive PBMCs in vitro over a given period of time? NB: the PBMCs/donors would not have "seen" the antigen before.
For example, if I took 1e6 PBMCs and stimulated them with a bacterial peptide pool (and perhaps additional co-stimulatory molecules like IL-2/6) for 5-7 days could I detect antigen-specific responses via indirect ELISA?
All the major players would present in the well; however, there's obviously no defined germinal center, etc so I have my doubts as to the validity of the whole exercise. Have any of you tried something similar?
Thanks in advance for your input!
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Hi Nathan,
Considering a restricted number of cells of a cell culture assay as you mentioned, it is unlikely that you stimulate the specific B cell clone to expand and differentiate into antibody-secreting cells.
However, other reagents (Pokeweed mitogen + Staphylococcus aureus cowan + CpG ODN) may present a better result if combined with a peptide pool for incubation with PBMCs.
The description of these reagents as an optimal stimulum for B cell expansion and diferentiation into antibody-secreting cells in vitro can be found in the paper below:
Tracking human antigen-specific memory B cells: a sensitive and generalized ELISPOT system. Journal of Immunological Methods
Volume 286, Issues 1–2, March 2004, Pages 111-122
Shane Crotty, Rachael D. Auberta, John Glidewell, Rafi Ahmed
Good luck,
Eduardo
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  • As we known, the naive B cell activation depend on T cell. The antigen alone cannot trigger B cell differentiation. However, whether the memory B cell could be activated by antigen without Th cell signaling?
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Actually, with certain antigens - ones that have a repetitive structure - primary B cells CAN be activated without T cell help. These antigens are known as T-independent antigens.
But memory B cells are absolutely dependent on T cell help. I spent several years as a post-doc working with Norman Klinman at Scripps on this precise topic. Look for a review by Phyllis-Jean Linton and Norman Klinman; she published that back in the late 80's early '90s. I do not remember the name of the journal.
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Hi All! I am looking for a CD20+ and CD19+ B cell line to use in upcoming work. My research has mainly led me to Ramos cells (Burkitt's lymphoma), but I'm unable to find information on the ATCC website that specifically confirms this cell line expresses CD20 and CD19. Can someone confirm if Ramos cells are the right way to go or if there is another cell line I should look into? Any help is much appreciated!
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Hello Lucia,
Ramos cells do express CD19 and CD20. You can have a look at the following publication (Fig. 1A):
CD20 as a gatekeeper of the resting state of human B cells
(Kläsener et al. 2021) Proceedings of the National Academy of Sciences Feb 2021, 118 (7) e2021342118;
DOI: 10.1073/pnas.2021342118
In the Materials and Methods section you can also find the catalog number of the Ramos cell line, which the authors used, and detailed protocols and cultivation conditions.
Kind regards,
Alexander
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Hello,
Can I use the B cells expansion kit after FACS sorting of specific B cells?
Because I read the protocol of the B cells expansion kit and they do it after B cell isolation kit using magnetic beads.
Thanks in advance.
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You may definitely use the B cells expansion kit on B cells isolated from either method (FACS or MACS). The Isolation method will not have any impact on the mode of action of stimulant.
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We are doing studies on B cell differentiation and would like to expand human B cells in vitro.
Thank you
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Thank you so much. That celline does not seem to be generally available. It also would be much better to have an adherent cell line so one does not transfer the sCD49L expressing cells with the growing B cells. Thanks again.
Thomas
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I couldn't find any anti-rat CD20 which has been tested for in vivo B cell depletion in rats. Could any anti-rat CD20 or CD-19 ab work for B cell depletion in rats?
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Dear Ines Simo
Without knowing the details of your experimental set-up, maybe it is worth evaluating B cell deficient rats recently published by Shannon Reese's group from the University of Wisconsin Madison.
All the best & kind regards,
Michael
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I have a basic immunology question that I am not able to find a definitive answer to so far.
B cells require activation by a T helper cells in order to turn into a memory B cell.
But is it necessary for the T helper cell and the B cell it activated to be specific to the same antigen?
My colleague says that CD40-CD40L and CD28-CD80 co-stimulations are not enough and describes a different process;
B cells must internalized the BCR-antigen complex, process the antigen and present it on an MHC-II molecule to a T helper. Only when a T cell is activated this way can it can activate the presenting B cell. Meaning, they must necessarily recognize the same antigen for the B cell activation and maturation to occur (of course, different epitopes in that molecule, but still the same protein antigen).
I was not taught that this step is critical; but that any stimulated and licensed T helper can provide the required co-stimulation to any activated B cell. Antigen-specific activation occurs stochastically, because both APC, T helpers and B cells are present in the same area when an antigen is incountered.
So far I can't find a clear answer for which of the two option is true, either in the basic or more specific literature.
Can anybody shed light on this, preferably with reference to proper sources?
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That makes sense.
Thank you for the input!
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Rituximab is a B-cell-depletion monoclonal antibody that is used to treat rheumatoid arthritis and other diseases. The chimeric structure of rituximab comprises human IgG 1 and kappa-chain constant regions and heavy- and light-chain variable regions from a murine antibody to CD20. Thus, I presume that rituximab could be used to deplete mouse CD20+ B cells. I also read some papers which use rituximab in mice before, but no figures about B cells after depletion were shown.
I used different doses of rituximab, and the drug was injected into C57BL/6 mice intravenously or intraperitoneally. One week after administration, blood, lymph nodes, and the spleen were collected and the number and percentage of CD19+B cells were evaluated. But no difference was observed between administrated and control groups.
Has anybody used rituximab to deplete mouse B cells before? Do I need to change a mouse CD20 mAbs rather than using rituximab if I want to deplete mice B cells?
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Dear Echo Zhu ,
I am citing a sentence from the below article:
>IDEC-C2B8 (=rituximab) is a chimeric MoAb containing human IgG1 (κ) heavy and light chain constant regions and murine variable regions from the murine anti-human CD20 MoAb IDEC-2B8 (murine IgG1, κ).
According to the sentence, rituximab derives from a mouse IgG1 directed against human CD20 but not against mouse CD20. So, I do not think you could presume that rituximab could be used to deplete mouse CD20+ B cells, though IDEC-C2B8 (=rituximab) and IDEC-2B8 might cross-react with mouse CD20.
If you wish to deplete mouse CD20+ cells, I could suggest the following product.
InVivoMAb anti-mouse CD20, clone MB20-11
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Hello Research Gate Community!
I am teaching a class on immunology for a general cell biology course, pretty much following the immunology chapter in Alberts (chapter 24). I was confused about a figure think I may have found a mistake in figure 24-47 but wanted to check with the community since my immunology background is not particularly strong. In the attached figure 24-47, on the right it shows antigen presentation between the effector-T-cell and B-cell. As I understand it, and as described in the legend, B cells bind antigen via their BCR, internalize and proteolyze it and and present it on MHC class II, which is recognized by the TCR on the T-cell. In the picture, I think the TCR-Antigen-MHC-II complex is upside down. The TCR (in black) should be down, attached to the T-Cell, right? And the MHC-II (green) should be above, on the B-Cell, right? I want to make sure I understand the process well for the class and would also report this mistake to the publishers. Thanks for your help!
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I totally agree with @Sayed Abdelwahab
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Hi,
I want to make the IsoFlow Sheath liquide in our lab by following the chemical composition given by the company ( mentioned down below) , i want to know if any of you tried this before and if it works without any risk during the aquisition process. and i would like to know also if there are any tips or precautions to take when preparing the solution.
Chemical composition for 10L :
Sodium Chloride......................7.93 g/L
Disodium EDTA........................0.38 g/L
Potassium Chloride.................0.40 g/L
Monosodium Phosphate.........0.19 g/L
Disodium Phosphate..............1.95 g/L
Sodium Fluoride......................0.30 g/L
Kind regards.
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IsoFlow in Beckman coulter or sheath fluid in BD machine are the isotonic buffers solutions which may be replaced by Autoclaved and filter sterilized PBS.
You should be extra cautious while using in-house sheath buffer as any particle or growth may block your tubes in flowcytometer.
We have used Autoclaved and filter sterilized PBS in BD instrument and it never caused any issue.
Once you use any in-house buffer, the parent company will consider it void of warranty. So, never let the company people know that your are not using their isoflow.
Good luck,
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I have faced multiple problems with both western blot and RT-PCR detection of cytidine deaminase in cell types other than B-cells. Can someone give me details of the detection and the details used (primers, antibody suppliers) or any technical tips you considered important
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Dear Armando,
This info below may help:
What kind of antibodies are used in western blotting?
Most commonly, the transferred protein is then probed with a combination of antibodies: one antibody specific to the protein of interest (primary antibody) and another antibody specific to the host species of the primary antibody (secondary antibody).
Overview of Western Blotting | Thermo Fisher Scientific - US
Can a western blot assay show multiple bands?
The Western blot assay is a powerful tool to study a protein of interest. However, if multiple bands appear in a Western Blotting, this may increase difficulties and troubles in the protein analysis.
Western Blot Troubleshooting Multiple Bands | Sino Biological
How to solve the problem of Western blotting?
Comprehensive solutions and suggestions are provided to help solve your particular western blotting challenges. To start troubleshooting your western blotting problems, choose the type of problem you are experiencing. Good western blotting results begin with high-quality protein electrophoresis results.
Troubleshooting Western Blots with the Western Blot Doctor ...
Which is the best blot enhancer for Western blot?
Use Thermo Scientific SuperSignal Western Blot Enhancer to reduce background and enhance detection of low-abundance and weakly immunoreactivity antigens. Increase the number of washes and/or the volume of buffer used. Add Tween 20 detergent to the wash buffer to a final concentration of 0.05%.
Western Blot Troubleshooting | Thermo Fisher Scientific - US
_____
Also, review the following links:
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Dear all,
Does anyone know if it's possible to do a bone marrow transplantation to Rag1-/- mice without preconditioning them with irradiation (donor mice are going to have the same MHC haplotype)? The idea is to reconstitute T and B cells in our double mutant mice (that were crossed to Rag1-/-) with WT GFP+ bone marrow cells.
Thanks!
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In rag1 and rag2 -/- human allogeneic hsct works without irradiation and conditioning regimen. So, it should work in mice, too.
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Hi!
I have to carry out a mouse B and T cell immune profiling FACS based assay from animals that have been immunized as a part of my study. I am new to immune profiling and I would be happy to get any pointers on building the antibody/ marker panel and the gating strategies would be very helpful. We have a BD facs verse.
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Dear Swetha,
As John Wilson just wrote, it all depends on what are you looking for.
Here are some more suggestions on where to look for gating strategies and markers:
- The OMIPs (although most are human) provide not only with the markers but the gating strategies and reagents : https://onlinelibrary.wiley.com/doi/toc/10.1002/(ISSN)1552-4930.OMIPscollection
- The EJI guidelines can provide you with the isolation protocol and gating strategies and also help with everything that is related with Flow cytometry :
- Some antibody companies like Miltenyi also have some guidelines on which markers to use for each populations:
I hope this helps.
Good Luck
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Hi has anyone used lipofectaimine for transfection of primary human B cells. Compared to Neon is it better or worse
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Thank-you Han Zhang ma'am for your reply
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Hi everyone ! I'm looking for an expression plasmid (hopefully with a fluorescent tag) for observing plasma membrane changes that work properly in B cells, this since I'm planning to acquire videos of this phenomena. I've found a lot of references for TULP family plasmids (TUBBY-EGFP for example) that works well in other cell models. Can you suggest any? Thank you in advance.
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CAAX-GFP is a great plasmid for this purpose
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We have recently seen a case of large diffuse B-cell lymphoma of probable folicular origin (CD10+ partial expressión) with a well defined CD103 expression by B-cells. The references in the literature are scanty and unclear.
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Jordi Juncà We have just analyzed the blood of a patient with previous diagnosis of follicular lymphoma in lymph node that returned to the hospital with adenopatias and abnormal lymphocytes in blood. We performed an immunophenotyping of the blood and found 12% of medium sized cells with characteristics of follicular lymphoma: CD19 weak, CD10++, BCL-2++, CD5 neg, CD43 neg and IgM neg. However, these cells showed some positivity for post-germinal markers such as CD27, CD11c and CD39. In addition, 50% of the abnormal population showed expression of CD103 (without expression of CD25, CD123 or LAIR-1). It is the first time I see CD103 expression in a follicular lymphoma. On the other hand, I´ve already seen cases of hairy cell leukemia (very classic) with CD10 expression.
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Our laboratory hopes to find a B cell line expressing PDL1, thank you!
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Also JeKo-1, Mino, Raji for human B cell lymphomas.
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Hi there,
I'm trying since more than 2 years now to perform a quite simple experiment that you easily find in the literature but it constantly fail whatever I tried !
So I purify magnetically (touch ou untouch I even FACS-sorted) splenic B cells from WT and my KO mice.
Then I coculture for 4 hours B cells with OP9 and OP9-mDLL1 cell line ( I also tried CHO/ CHO-mDLL1 and even transiently transfect CHO to increase mDLL1 expression).
My cell lines are mycoplasma free.
After the 4 hours coculture I recover the B cells, purify RNA with a kit, reverse transcribe and make a qPCR on Notch target genes HES1 and DTX1 (and housekeeping gene of course).
In theory DTX1 and HES1 expression should strongly increase on WT B cell cocultured with DLL1 but in my case expression doesn't increase at all compare to cells cultured without DLL1, same with the KO B cells.
So it's like the signal is not given and NOTCH2 not activated on B cells!
I tried to extend incubation time 6h or overnight
I tried to put total splenocytes
I tried to coculture the cells under centrifugation 37 degrees (30 first minutes or 2h then incubator for the rest of the time to reach 4h) to force them to interact.
I tried to coculture the cells in 96 wells flat and U-bottom or in larger wells.
Same results!
I don't know... Is there anyone here or knows someone who do or did this kind of experiment and can help ?
Thanks a lot in advance for your help !
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Hi Sarah,
Nope because I assume that 4h of culture is not enough for my B cells to die...
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I sorted the B cells then stimulated them with CpG and cultured for 6 days but i can see a contamination from day2, i cannot used antibiotic for this experiment because its a control for my experiment, how can i avoid this problem.?
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Ensure the cells are taken from the patient using asceptic technique. Carry out the cell sorting procedure under asceptic conditions as far as is possible. Primary cell cultures are at the greatest risk of contamination as usually antibiotics ,anti bacterial as well as anti fungal are included. However they can be omitted later on as the cells become adapted or immortalise.
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I have a question about rabbit GC B cell. I am working with respect to virus and how it affects rabbit GC B cells. However, i am unable to get any good antibody for GC B cells. Tried BCL6 but the clone i selected is not staining well. I was hoping to get some answers here.
Thanks in advance.
Naren
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Thank you very much Lora Benoit . I tried PNA from vector labs and unfortunately its staining for T cells and am also getting non-specific staining.
Any other GC markers reactive with rabbits would be helpful.
Thank you in advance.
Naren
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I'd like to evaluate T-cell dependent B cell responses in PBMCs without isolating T and B cells. Does anyone have a protocol for this type of assay? Could I culture PBMCs with anti-CD3 and anti-CD28 beads to activate the T cells over a number of days and then looks at B cell responses by flow cytometry and soluble IgG in the media? Any ideas?
Thanks,
Penny Anders
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Thanks Alexandr,
The article focuses more on T cell activation. I would like to activate T cells but then look at T-cell dependent B cell responses.
Penny
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I'm trying to make a co-culture with AGS cells and B cells, and we want to do a sorting. Does anyone here know a good surface marker? this cell line doesn't express catenin beta 1, e-cadherin and vimentin
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You welcome!
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Hi!! Do anyone know if CD8+ T cells are affected by CpG in a culture? Because I want to coculture B cells and CD8+T cells, so I have to activate B cells with CpG; but what about CD8+T cells and CpG?
Thanks!!
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Dear Yamila Gazzoni Yes, CpG does effect the clonal expansion of CD8 cytotoxic T cells. I hope the publication attached will be helpful in answering your query as the stimulants used in B cell culture (B Reg differentiation).
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I have been recently working on JM1 cell line which is said to be a premature B-cell but is unable to understand what is used to activate the cell line. It is already said in ATCC website that this lacks IgM, hence I am unable to understand what can be a specific and a nonspecific activator of the cell line will be.
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Dear Kaustav!
May be You look at the following article:
Table 1
Blood cancer cell lines carry nonproductive BCR/TCR in either both alleles or the only expressed allele
B cell lymphoma unspecified JM1
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Hey, guys,
I thought it is reseonable to speculate that if one PI3K inhibitor can reduce p-ERK, pAKT or p-S6 more dramatically in cell line A compare to cell line B (A cells were crispr knockouted one gene, and B cells is the control), this drug is more sensitive in A than B, given the fact that the basal level of phospho protein is higher in A and A cell is also grows quicker. Dose anyone has experience on this? is cell proliferation the only way to check drug sensitivity?
Thanks
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Hey, John, actually you kinda replied my second comment. so yeah, p-protein should not be used as biomarker in this regard. I have see one report claimed that p-ERK had no relationship to proliferation inhibition of MEK inhibitor trametinib. But reduced p-ERK can sensitize other drug for cell growth inhibition.
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A very important study from Denmark published yesterday in the Lancet on 4 million people focused on reinfection of sars-cov-2.
The protection rate for those who have been infected is 80% for adult, and 47% for those > 65 in age.
It means there are 20% and 50% chance of reinfection in adult and older, respectively.
What could be the possible explanation? Immune response differences, CD4, B cells, and TH17 efficacy.
Do we have to reoptimize vaccine guidelines for whom have been previously infected?
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Immunosenescence as the root cause of older people at increased risk of COVID-19 infection, reinfection, outcome, and response to vaccination
It is well known that older people have immunosenescence that predisposes older adults not only to SARS-COV-2 infection, but also reinfection and poor outcomes. Even the vaccines have poor response in the older people. Strategies are being developed how to tackle Immunosenescence to reduce infection risk and improve response to COVID-19 vaccine (1-4).
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Please find attached the full medical file of our patient and all biological reseaults included.
We are seeking to explain how can she produce such levels of immunoglobulins with no B cells expression.
NB ! : * The patient ddidn't recieve any immunoglobulins injection.
* We made another dosage for immunoglobulins levels after 01 moth of the first one and we had the same resault ( high levels ).
Best regards
Msc, Abdelwahab
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First thing i would like to comment on it that you may change the clone of CD20, check CD19 as well if they are even few?
Secondary, The absence B cells might be the drug effect (Like: Rituximab or any alternative/traditional medicine).
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Hi,
Does anyone know of a protocol to increase the TOTAL number of naïve CD19+ B cells in culture from a total count of 105 to a total count of 1015 , please ?
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Try using LPS 10-50 ug/ml, B cells expand vigorously.
and the other commonly used is anti-CD40 with/ without IL-4.
However LPS seems to sensitize the B cells towards apoptosis, and survival rates are better for anti-CD40.
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This is a follow-up on an earlier question regarding mechanistics of vaccines: why current vaccines don't block transmission. In summary, they don't engage mucosal immune cells to produce IgA. Thus, they induce production of IgG by systemic B cells and can lower viral load, but not block viral shedding or uptake through mucous membranes. "Herd immunity" is dependent on halting transmission. So, the AstraZeneca trials were the only ones that assessed sterilizing immunity. Their surrogate for transmission was asymptomatic cases. They found that their LD/SD regimen was about 60% effective in blocking new asymptomatic cases. My first question, then: was this simply due to a reduced viral load (and reduced coughing perhaps) which was expressed as a lower transmission rate? Can this even be equated with measles-type herd immunity? Would this 60% effectiveness at blocking asymptomatics on the basis of reduced viral load be sufficient to achieve herd immunity?
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Hi Michael and Lora.
As a mucosal immunologist I like to add to this interesting discussion. For local IgA production (i.e. IgA produced in the mucosa in the oral cavity) it is generally thought that you need to see the antigen at the mucosa or related lymphoid organs - in this case likely the tonsills - for production of antigen-specific IgA-producing cells to home to the mucosa to become plasma cells. In genel it is not likely that this will happen when people are vaccinated with a novel vaccine systemically. However, mucosal homing may happen if you have had a disease before, and then boost systemically (i.e. the vaccine in people who had the disease). Since some recent papers suggest cross-reactivity of antibodies between SARS-CoV-2 and other coronavirus in the early response, it is possible that we will have cross reactive memory B cells formed at the mucosa that may home to the mucosa after vaccination with the systemic vaccines. Also remember, even if you see IgA in serum, this is likely from IgA producing cells in the bone marrow, and is not the same IgA that is produced at mucosal surfaces.
A different question is whether IgG (or IgA) in serum is sufficient to block infection to give sterile immunity - and also if systemically primed T cells play a role in fighting infections at the mucosa. I am not a virologist, but the general thinking is that IgG and T cells may hinder mucosal infections but not totally block them. There is, for example, often a rather good correlation between IgG levels in serum and protection for many diseases.
Nevertheless, IgG and T cells may still decrease the likelihood for infecting others, as this is dependent both on local viral loads at the mucosa and symptoms (sneezing and coughing spreads virus). Thus, it is likely that the vaccine will diminish infection rates even if there is no local IgA production. Similarly, if the virus mutates, even at the spike, this will likely decrease protection, but not total lack of it due to IgG binding other sites or T cell reacting to T cell epitopes.
Again, please remember that I am not a hard core vaccinologist or infection virologist studying coronviruses as my speciality, but my response would reflect the general thinking among most mucosal immunologist around the subject.
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I know that efficiency of transfection for primary B cells is very low with Lipofectamine. But I did not try that for EBV immortalized B cells.
Do you have any experience with transfection of EBV immortalized B cells with Lipofectamine?
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Dear S. Nakazawa!
You look at following articles, please:
Cell growth and matrix invasion of EBV-immortalized human B lymphocytes is regulated by expression of av integrins
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PSSM(Position-specific scoring matrix) is one of the key features to be used for B cell conformation epitope prediction but I am confused about how to use it as a feature.
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You can use : PFeature
go to the evolutionary info.
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We have some human cells (from the tissue of a cancer patient) growing and proliferating in suspension for more than a year now. The medium is RPMI-1640 with L-glutamine, P/S and 10% FBS. No extra goodies added.
Flow cytometry: Most of the cells are CD19/CD20/CD45/CD81 positive. Some express CD138/CD27/CD28/CD73. And there are human IgG1 in the culture supernatants. It seems that they are B cells and plasma cells. But are these cells immortalized or malignant (myeloma) ?
I also did the Wright stain (see the attached PDF file), it seems that there are cells of variable size or cells at different stages or cell debris? We have very limited experience on B cells in the lab and really do not know much about the morphology.
Thanks for any inputs!
Lixin
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Yes, the cells are positive for EBV (type 1). Also the LMP1 may have the 30-bp deletion at the C-terminal.
Lixin
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Such a thing should be hypothetically possible as B-Cells like all other nucleated cells possess MHC-I, & might be able to cross present extracellular antigens similar to other professional APC's via MHC-I to cognate CD8+ T-cells. Does this process lead to the cytolytic killing of the antigen presenting B-cell by cognate CD8+ effector/central memory/antigen primed T-cells in the lymph node? (If so what are it's implications w.r.t the adaptive immune response?)
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It is a normal part of the contraction phase of the immune response
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I am trying to isolate B-cells from the spleen of a STAT3 KO mouse and plan to stain with Annexin V and 7-AAD. I will then use flow to determine apoptosis and overall cell death. I have used Staurosporine in the past; however there was no distinct difference between the cells that were treated with Staurosporine, LPS, and non-treated. I am trying to figure out if it is the problem is the timeline, the treatment, or the staurosporine. Would love some help!
Thank you!
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In case of Staurosporine, maybe the concentration you employed is higher. Usually, doses lower than IC50 will be better for special incubation time.Also, for apoptosis, you can use mitomycin c and 1% H2O2.
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It is known that scientific knowledge is replicated each time at more unusual speeds, however, humanity expects more from our scientific work, that is, solutions to problems that have accumulated and that it is necessary to solve to improve the quality of life . It would be good to reflect on those expectations.
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The experience of the development of science shows that the quality and significance of a scientific discovery and its usefulness or harm for human civilization are inversely dependent on any expectation, be it the expectations of the entire scientific community, and even more so the expectations of a "non-professional" society. For example, it is known that the great Rutherford, who discovered the atomic nucleus, believed that his discovery would have no practical application.
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our team is looking for a tecnic bases on culture b cells from cirrhotic patients, and ACLF, in his own plasma and also do a crosslink betwen other plasma from healhy controls, please do you know some other groups that do the same, or with others ilness? Thank you.
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Isolated B-cells were stimulated with anti-CD40 antibodies and TLR9 agonist to assess costimulation marker expression, cytokine production, immunoglobulin production, and CD4+ T-cell allostimulatory capacity.
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I want to evaluate B cell activation by flow cytometry using B cells in human peripheral blood.
I have difficulty in establishing a positive control.
What is the best stimulator? Lipopolysaccharides, B-cell Activating factor, or other reagents?
What kind of markers are necessary for evaluating activation by flow cytometry?
CD19, CD27, CD24, CD38, IgD and IgM are enough for it?
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For B cell proliferation, cells were stimulated with anti-IgM (10 μg/ml, Jackson Lab; 109–006-129), CD40L (100 ng/ml, Enzo; ALX522–110-C010), CpG (0.5 μM, Enzo; ALX 746–006-C100), IL-4 (50 ng/ml, Peprotech; 200–04), and IL-21 (50 ng/ml, Peprotech; 200–21) as indicated in the figure. After incubation for 3–5 days (3 days for T cells and 4–5 days for B cells), cells were stained with fluorochrome-conjugated CD4, CD8, or CD19 (BD Biosciences, Clone RPA-T4, RPA-T8, HIB19 respectively) for 30 min at 4 °C (dark). Cells were washed with PBS twice, and cells were acquired and analyzed by flow cytometry (Becton Dickinson FACSCanto II) and FlowJo software (FlowJo 10.5.2, TreeStar).
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Dear scholars,
Our research is currently involved with hybridoma technology. We have isolated some original clones after fusion and we are preparing the subclone step to find some true positive clones. After subcloning by limited dilution (LD), we started to store the remaining original hybridomas into liquid nitrogen (2-5x106 cells/vial). After a month in liquid nitrogen, we cannot find any positive clones in the subclone stock. The supernatant we collected after thawing and culturing for 3 days from the original clone became negative compared to the supernatant before storing in liquid nitrogen. We tried to thaw and re-do the LD again but still have not found any positive clones.
+ We used PEG to induce fusion between myelomas and B-cells
+ We used indirect ELISA to screen for positive clones
+ We upscaled the positive original clone to 100mm culture dish (SPL Life Sciences) before subcloning by LD and store in liquid nitrogen.
+ We used NutriFreez® D10 Cryopreservation Medium to store hybridoma cells in liquid nitrogen (We stored the cells in a container box (Nalgene® Mr. Frosty) at -80oC at least 4 hours before moving them into -196oC in liquid nitrogen)
Has anyone encountered this situation before? Please let me know and I am very thankful for your answers.
Sincerely.
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Hi Cuong,
Once your hybridomas are thawed and grow exponentially, do they stop secreting antibody? Or do they secrete an antibody that doesn't recognize your antigen?
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Hi, guys,
Do you have problem using B cell negative selection kit form Meltinyi?
Sometime the B cell is pure, CD19+>95%, but sometimes it lower than 90%.
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Thank you.
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Hello,
I am reading on vaccines in general and I wonder if the beneficial effects of vaccines (as vaccination is the most effective choice when pathogens are to be stopped and prevented) lower the Darwinian fitness of immune cells such as T-cells and B-cells.
Thank you for your answers!
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may be
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I am analyzing my cells of interest, such as B cells, when i apply gates from one sample to another, am facing the applied gate in the second sample seemed that it is missing some populations. Such as, in sample 1 the CD27+ CD21+ has 10,000 the level of expression and second sample the maximum expression is about 8000, thus when i apply the first gate to the second, i think i would miss some dim population in the second sample. FYI, the voltage for these sample is similar during acquisition. Further, am also seeing this even with sample which were stained together at the same time. Therefore, should leave the second sample as it is or move to include all CD27+CD21+ cells?
I will look forward for your assistance
Thank you very Much!!!!
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Hi,
Different individuals can have different expression and "shape" to the populations.
You must adjust gates between different samples, and your control needs to be cells from the same sample/individual, but without the marker in question (FMO).
Kerstin
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I was posed a question by a hematopathologist in training this morning: Why are B-cell lymphomas more prevalent than T-cell Lymphomas?
My initial guess was perhaps there is increased tolerance training in T-cell populations, but it turned out she did not know either. Her best guess was that it is multifactorial in nature, i.e. genetics, viral infections (HTLV), and environmental factors.
Does anyone have any papers or book chapters for reference? I would love to be able to bring a more detailed and concise answer back to her. So far, I haven't found anything that answers the question on PubMed.
Thanks in advance.
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Development, activation and contraction is more tightly regulated in T cells v B cells. Particularly the latter, so you are less likely to see t cell lymphoma secondary to chronic immune stimulation for example malt lymphoma. Also, ebv. B cells express cd21 whereas t cells only transiently do so during thymus development. So ebv causes hl, bl, mzl, malt, and dlcbl in b cells but basically, only extranodal nk/t cell lypmphomas in the cells as I recall. 97% of adults are ebv seropositive.dlbcl is the most common lymphoma in the us, roughly 15% is eber+.
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I purified some ~2x10E8 B-cells using magnet separation, and since I am not using it until the end of the treatment of my animals, I tried to freeze it.
However, after centrifuge them for 10 min at 4oC at 200x and later at 800x as someone suggested me it didn't form a pellet at all.
Can someone give a suggestion?
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Thank you very much Dr. Chernov, I find out that this just happened to the control samples. Hence, I my senior recommended to divide these 11 mL samples in a 10.5 mL upper fraction and left the 0.5 mL in the original tube and count the cells. As I counted the cells I found around 10E7 cells in the later and 10E5 in the former. Thank you for your help.
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Dear All,
I am wondering if you know of any gating strategies for analyzing T and B cell populations in Murine blood samples collected in heparin-coated tubes.
As far as I know,
100ul of blood, stained with appropriate antibodies should be lysed, fixed, and subjected for analysis.
For your information, I have Four color FACS machine and would like to know the antibody panels used for analysis along with the gating strategies would be a great help.
For instance, I was thinking of the following panel, CD3, CD4, CD8, B220- for the T cell population
and CD3-, B220+, CD19+ for B cell population.
Please enlighten me if I am wrong.
Thanks in advance.
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Antigens are processed by specialized macrophages to produce complex protein-RNA complexes that eventually produce iRNA. When this iRNA is introduced to primary B cells that have never seen the antigen, they produce specific antibodies to that antigen. Th macrophage produced RNA is incorporated into the the genome of these B cells by reverse transcriptase that now become "memory cells" capable of a secondary response when confronted with the antigen they had never come into contact before. Reverse ttranslation in the macrophage is the best explanation for the production of such specific iRNA.
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Yunus Shukor Thanks.
Indeed between a choreographed dance of hypermutations and recombination, gene splicing and translocation of gene segments to generate an enormous variety of VxDxJxC expressions for an antibody or the existence of a reverse translatase that would process the exposed epitope of an antigen and encode an RNA segment for the hyper variable segment of an antibody, nature and Occam‘s Razor I feel would pick the latter.
Thanks you M. Y. Shukor.
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I am trying to see if any ERK1/2 translocation into nucleus in B cells after activation but couldn't see anything so far. These are the activators that I used;
- F(ab')2-Goat anti-Human IgG, IgM (H+L) Secondary Antibody, Functional Grade (Invitrogen)
- Animal-Free Recombinant Human sCD40 Ligand (Peprotech)
Basically, I collect fresh PBMC from healthy donors and prepare them to seeding onto 96-well plates (washing w/ 1X PBS, counting etc.). Seeding 200K cells/well in 200ul cRPMI, activating cells usually 60 mins but in the last experiment I've changed activation time as 5-10-30-60 mins. 5 mins was the best of them for ERK staining, yet I have no signal for pERK. Antibodies are listed below
- p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb (CST)
- Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb (CST)
- AF488 secondary Ab (CST)
I've done all the experiments what CST says in their protocol here: https://www.cellsignal.com/product/include/protocol.jsp?productId=4695&protocolId=12326242&print=true
Also I'm attaching ERK vs pERK staining in 5 mins activated cells, as you can see there is nothing in pERK. I was thinking that I still may not be able to penetrate nucleus but also open to any recommendations.
Thanks in advance.
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Why don't you try for time gradient by Flowcytometry. It will save your time as well as efforts.
Flowcytometry will also be good in determining wether the Ab is penetrated to nuclear compartment if you run a tube for secondary alone.
Once you get results, you should shift on to microscopy.
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The idea is show drug of interest is not affecting functional ability of these
I'm thinking along the lines of:
1. Stimulate primary macrophages (or just Raw 264.7 cells) with LPS and treat with drug of interest. Label the macrophages and run on flowcytometry.
It doesn't sound right to just do this or may be I'll have to test for engulfment of FITC zymosan beads etc as the idea is to test the ability. Any thoughts?
2. Activate B cells with LPS/IL4 and treat with drug of interest, Measure IgG1? I'm not sure if this is called quicker as minimum activation period is 12-24 hrs. Any other way? IgM would be quicker ?
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For macrophages you may perform phagocytosis assay using fluorescent labelled E. coli or S. aurious, which takes around 60-80 minutes and check on flowcytometry.
Or you can check oxidative burst by Flowcytometry, it also takes around 45-60 minutes.
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Hello Everyone.
Greetings,
I am planning to perform short term B cell culture (without any stimulation) from ovarian cancer patient (source of B cell PBMC and Ascites). I don't understand how long I should culture them. I will check the cytokines and Igs secretion by the B cell. Can anyone please suggest any protocol for short term B cell culture or any reference paper that can help me in my research. Thank you in advance.
Sincerely,
Razaul Haque
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It all depends on what you like to measure. If you are looking for cytokines or antibodies from resting memory or naive cells I would doubt that you would get enough for meaningful measurements. However, incubation of unstimulated PBMC for 2-3 days is actually a quite good way of looking the antibodies produced by ongoing plasmablast (or in tissue plasm cell) responses. In this case, the PBMC are left in media and supernatants are collected. These are subsequently used in ELISA or other assays for detection of antigen-specific (or non-specific) responses. See for example:
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Recently I performed RBC lysis to obtain WBCs from human whole blood. For my application, I use immunofluorescence to identify neutrophils and lymphocytes from this WBC population. The antibodies I use are CD3 (T cells) and CD19 (B cells) for lymphocytes and CD16 for neutrophils. I observed non-specific antibody binding, for example, lymphocytes were staining for CD16. Also the expression levels of all the antibodies were quite low. When I use density-gradient media such as Polymorphprep for separations, I don't face these issues. I am wondering whether there is evidence to say that lysis might be the cause here and if so, I would love to know why.
Any insights here would be greatly appreciated.
~Ani
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In humans, CD16 is present on peripheral blood natural killer cells which are lymphocytes and will appear in the Lymph gate. They are CD16+, CD56+. The marker is also present on some monocytes (usually CD14) and may indicate activated monocytes
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I have multiple epitopes that are potential binders to both MHC-I and MHC-II and they also have B-cell inducing capacity (All predictions are made through immunoinformatics). Now can anyone suggest a linker sequence to join these peptides which don't affect any of the three responses? I have consulted the literature which says GPGPG linker is suitable for HTL epitopes but acts as an antagonist for HTL responses, whereas AAY is suitable for CTL epitopes and vice versa. Now can anyone have any suggestions?
Thank you!
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Thank you Saroj Kumar Khan for your suggestion!
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Hi,
What is the common B cell differentiation stage used for hybridoma technology? I understand that these are antibody producing B cells upon the injection of antigen, but do people use mature B cells or fully differentiated plasma cells to fuse with myeloma cells?
Or is the specific cell differentiation stage less of a concern?
Best,
Cece
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Early B cell stage is not useful as a fusion partner on hybridoma technology. Actually please do not prefer naive mature B cell ..You have to wait Ig class switching after immunization for good antibody production (Ig class switching needs T cell help). If you use early stage B cell (immature or naive mature B cell), yo can get just low affinity IgM.
Sincerely
IShak
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I performed flow analysis in the mouse spleen (Wild type BL6 mouse), and found the CD79a expression is very low (~1.38% within CD45+ gate). But other B cell marker CD19, B220, CD79b are normal (54.8%, 52.2%, 46.2% respectively within CD45+ gate). I couldn't understand why CD79a expression is quite low, and I got the similar result even I increase the CD79a antibody concentration.Please see the attached image for gating strategy and B cell marker proportion. Can anybody explain this? Thanks a lot.
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Your experiment and compensation seem absolutely fine. It's omnipresent marker for B cells, so it should show as much as B cells (52-54%).
I would suggest you to do an experiment with normal human blood ( if your antibody has reactivity to human, I guess it should have). Even if you don't get the similar % of CD79a as to CD19, I would suggest you should write to the application specialist of provider, as it is a technical issue (possibility of cold chain break during supply or multiple freeze-thaw or exposure to light).
Clone may be an issue, it Will also be ruled out by doing experiment on different species.
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I would like to try activating B cells (or B cell lymphomas) by adding an anti -IgM antibody. Does anyone have experience in doing so? Should I rather use a whole antibody or (the more expensive) F(ab)2 fragments? Which concentration should I try for a start?
Thank you!
Anna
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I offer another vote of confidence for the F(ab')2 anti-mu reagent. 10ug/mL is also a good place to start, but it depends on your intended experimental output. Are you looking to evaluate proximal events downstream of BCR ligation (phosphorylation or Ca2+ flux?), upregulation of surface markers (CD80/86, MHC II), or maybe something else?
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Does anyone have experience of extracting DNA out of dairy cow's milk? I am interested in the DNA present in the B-cells. I have done extraction from blood but want to try extraction from milk. I have tried centrifuging the milk i) 2000 g, 20 minute, 4 C, or ii) 5000 g, 30 mins, 4 C in a falcon tube. I find the fat layer on top very difficult to get rid of and will contaminate the pellet at the bottom even after washing with PBS. Additionally, there are debris contamination in the pellets. The DNA yield from the pellet were not good and the target gene was not seen in the PCR amplification process. I used commercial spin-column based DNA extraction kit.
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New Rapid Method of DNA Isolation from Milk Somatic Cells https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4806335/
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We have a t-cell and b-cell assay we are working on, and found from customers they want FFPE as a sample type. I guess what we are trying to understand, while we are waiting on marketing, is why would customers not just use blood? Is it because the patient is no longer available to get a blood sample so they use the biopsy? We basically have been told if we don't include FFPE as a sample type, they don't want it.
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Thank you Michael, our assay is a Capillary Electrophoresis based assay. We did get more info that we were on the right track, sometimes the biopsy doesn't give the pathologist conclusive results, so they cut a few more curls from the FFPE block and send it off for processing. We have gotten all we need to get started at the moment. :)
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I want to check the CD19 marker on the surface of the mouse spleen B cell by flow cytometry. What secondary antibody can I choose that does not have non-specific connections?
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Very first thing, why you want to purchase an antibody without fluorescence tag. Go for a fluorescence tag antibody so that you need not to do indirect staining and 1 extra step.
If you have an unconjugated antibody with you then you need to buy the secondary antibody compatible to primary. Usually on the technical data sheet, you will find the compatible suggestions.
To check the nonspecific binding of secondary antibody you should run experiment;
1 tube of unstained, 1 tube for secondary only (to check non specific binding) and last 1 for primary antibody staining followed by secondary.
Hope this is usefull.
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What type of medium do you culture your B cells in (especially the FBS )? How do you stimulate them with BAFF and CdG or with IL?
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We culture B cells in RPMI 1640 (with L-glutamine), 5% human serum, 10 mM HEPES and 1X antibiotics with required stimulants (i.e IL2 or IL4) for our experimets.