Questions related to B Cells
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?
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?
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?
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
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?
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
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?
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,
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.
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.
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!!
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.
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
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!
- 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?
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!
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.
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?
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?
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?
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!
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
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
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.
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.
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.
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.
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.
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 !
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.?
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.
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?
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.
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?
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?
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 ).
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 ?
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?
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?
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.
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!
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?)
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!
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.
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.
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?
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.
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%.
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!
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!!!!
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.
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?
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.
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.
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.
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 ?
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.
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.
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?
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?
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.
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?
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.
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.