B Cells - Science topic

Hi Christoph Matti

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

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:

This seems like an exam or homework question for a class.

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.

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.

Hepatocytes in the liver express and secrete a great deal of protein, including serum albumin, which is the most abundant protein in blood plasma.

Fabian Flores-Borja Thank you for the reply!

Yousef Shahwan

"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...

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.

Hi Christina Vogelaar

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

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

Thank you Alibek, I will do it and I hope it will work :)

Hello Kamesh Ravichandran

You may use C57BL/6 mice.

Please refer to the two articles attached below.

Best.

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.

I think, u can check out the irradiation dose released by the machine, using dosimetric film?

A couple of years later... I am facing the same doubts! It would be great if Louisa or Alexander could send me those papers!! ;)

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

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.

Hello Lucia,

Ramos cells do express CD19 and CD20. You can have a look at the following publication (Fig. 1A):

(Kläsener et al. 2021) Proceedings of the National Academy of Sciences Feb 2021, 118 (7) e2021342118;

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

Dear Amina Abdelhadi ,

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.

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

That makes sense.

Thank you for the input!

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

I totally agree with @Sayed Abdelwahab

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,

https://www.sinobiological.com/category/wb-troubleshooting-multiple-bands

In rag1 and rag2 -/- human allogeneic hsct works without irradiation and conditioning regimen. So, it should work in mice, too.

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

Thank-you Han Zhang ma'am for your reply

CAAX-GFP is a great plasmid for this purpose

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.

Also JeKo-1, Mino, Raji for human B cell lymphomas.

Hi Sarah,

Nope because I assume that 4h of culture is not enough for my B cells to die...

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.

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

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

You welcome!

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).

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

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.

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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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).

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.

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.

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

https://www.nature.com/articles/1203509.pdf?origin=ppub (attached)

You can use : PFeature

go to the evolutionary info.

Lindsey Crawford

Yes, the cells are positive for EBV (type 1). Also the LMP1 may have the 30-bp deletion at the C-terminal.

Lixin

It is a normal part of the contraction phase of the immune response

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.

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.

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.

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).

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?

Thank you.

may be

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

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+.

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.

Here it is.

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.

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.

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.

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:

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

Thank you Saroj Kumar Khan for your suggestion!

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

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.

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?

New Rapid Method of DNA Isolation from Milk Somatic Cells https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4806335/

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. :)

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.

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.