Immunology Protocols

Most previous suggestions look appropriate. If ever you use negative magnetic separation after density gradient (getting rid of all other PBMC subsets with lineage MAbs) take care to work at 4°C. In my past experience using Dynabeads, I realized that monocytes "like" beads in general (since they are opsonization-specialized cells) and they tend to engulf the beads quite non specifically. Then, you may loose quite a few during negative selection. Room Temperature is enough for that, so keep them cool ! I would not recommend using positive magnetic sorting (e.g. with CD14) since your monocytes may be full of ingested beads after the procedure and they also can have "bad digestion", as human beings eating stones. Always question which level of purity your experiment is needing. The recipie will differ enormously from 80% to 95% and even more significantly for 99%. Then you electronic cell-sorting (flow cytometry) can provide high purity but with a low recovery and high cost/efforts. Last, you may associate several technologies: density gradient, ii) negative enrichment (in the cold) and finally elect. cell-sorting. Good luck. PP P.S. James, could you give more on your (very interesting and new - for me) statement: "CD14 can be expressed on cells other than monocytes,"

A few suggestions. We routinely maintain MCMV and plaque on NIH3T3 cells with no problems, but there are several things to consider. First, you should maintain your cells on a strict 3T3 regimen until setting up this assay- this means seeding 3x105 cells into a new T75 every 3 days- never allowing them to become confluent (if you are using T 150s seed 6x105 cells). By doing this you retain the cell charateristics more consistently. Next, we seed 12 well plates with 1x105 cells per well 24 hours before setting up the plaque assay. We dilute our virus in regular 3T3 medium (serial 10 fold dilutions), remove the media from each well adding first 100 ul of normal 3T3 medium and then 100 ul of our virus dilutions in duplicate wells. After incubation at 37 C and rocking every 10 minutes for 1 hour we remove the inoculum and replace with overlay: a fresh 1:1 mixture of 1% agarose (sterile in water- melted and equilibrated to 42-45 C) and 2x DMEM with 10% BCS (so final is 0.5% agar, 5% BCS- we make the 2X DMEM from powder). We incubate 5 days, at which time the plaques are easily seen. We then fix the cells by adding 10% buffered formalin directly onto the overlay (fill up the well) leave at least 24 hours to allow the formaldehyde to difuse and fix the cells, then remove the overlay and rinse. After airdrying you can stain with 1% crystal violet and the plaques are easily seen as clear areas surrounded by some darker cells, as the infected cells stain more intensely. If you have some smaller plaques, these may lack the clearing, but will still have more intense staining and should be clearly visible, with CPE with your 10x objective if you want to confirm.

Hi Mia, you could also try the FACS Jazz from BD Biosciences. http://www.bdbiosciences.com/eu/instruments/facsjazz/index.jsp?WT.srch=1&gclid=CLmTuqyUqbcCFbQetAodehsAQA Bert

I think still lowest DeNV2 titer. For that reason, you must harvest on flask (for 2-3 times blind passage or until CPE occur) to get highest titer then you can do plaque assay. Thank's

Sorry to those following the thread. Ben and I were just chatting and deleted our conversation as it doesn't enhance this thread.

Hi, can you perhaps give some details? Media and serum additives as well as flask characteristics all influence cell attachment and growth.

When I used to do this, we would release the bound cells with a scraper, or a low pH wash [3 ml of RPMI 1640 pH=3.0, ice cold, adjusted with acetic acid], collected into 12 ml D-PBS at pH 7.4. The brief exposure to low pH will dissociate the bound antibody from the cells and is not toxic, so long as you don't keep the cells in the low pH very long.

You can use the QuickZyme Total Collagen Assay which is marketed by Quick zyme Bioscienes. This assay is based on colorimetric or HPLC based detection of hyroxyproline.

You could also try to combine LPS with IFNgamma, this combonation enhances the production of pro-inflammatory mediators, due to the fact that IFNgamma is a coactivator of the LPS response. Please have a look in my publication from 2012...

Agreed with Abdelhalim Boukaba's suggestion. You should strip your membrane if you want to incubate another primary antibody.

I would also suggest not to reuse the beads. I tried ones but the purity was very poor. i end up throwing away all cells and simply waste of time..do not do this..

Hi Thang, the problem is, if you detect with an antibody it's not quantitative anymore. The big advantage of radioactive labelling is its sensitivity and it allows quantitation via a scintillation counter (-> for determining the Kd value). The amount of 35S-methionin that is used in such assays is very little, and 35-S is also not so critical as other isotopes because it is easy to shield (its mainly beta irradiation) - but of course you have to have a special training to handle radioactive material. You may google if there's an alternative labelling technique available (via luminescence probably?) which allows a quantitative readout. Best wishes, Andreas

Hi Cristina, if your experiment were a flow cytometry based proliferation assay then you would not need to start with a 'pure' Treg population, unless you were worried about other cell interactions, it could save quite a bit of hassle. You could separate your Treg target population based on combinations of the markers suggested in several of the other answers. If for example you were to perform a CFSE proliferation assay then you would also be able to stain for phenotypic surface markers and also permeabilize the cells for intracellular markers such as FoxP3 etc at the end of the assay before running the samples, gate Tregs in and non Tregs out etc. in the analysis. As for the combinations of markers, it might be a case of trial and error to see what actually works best for your samples. Hope this helped, Rachel.

Working with my particular strain of exosomes (DiCAPs), I have found it extremely hard to get them to attach to glass. Possibly, this has to do with their hydrophobicity. In the end I have resorted to heat treatment, by repeated (up to 9x) conduction through a bunsen phlame. Surprisingly, this treatment has worked well and allowed immunohistochemistry. Regards Thor

Good luck in your studies, Maria!

As I said, we use the PBMC (for functional experiments, so PHA stim would not be appropriate); my question is specifically about isolating DNA from the bottom of a Ficoll gradient. I know that this can be done but would like protocol advice from anyone who has tried it.

Yes, you could read the plate at 650nm and at the other wavelength and then, to make manually the corrections in Excel, but you must do the corrections in both: your samples and the standard concentrations curve. If even so, resulting concentrations aren’t logical for you, then I advise you to do the test again.

The isotype control serves two purposes. Firstly, It would confirm the specificity of your primary antibody. Secondly, it will show you the fluorescence resulting from FC receptor mediated biding and other non-specific cellular protein interactions. The isotype of an immunoglobulin (antibody) is determined by the differences of amino acid sequences in the constant region (FC region) of antibody heavy chains. So, when you consider the structure of your fluorochrome -conjugated primary antibody, it is composed of a FC region that belongs to the host species where the primary antibody has been raised. So, the isotype of your primary antibody is determined by the host species. The variable region is able to recognize the particular antigen. What happens in a FACS staining when you incubate your cells with the primary antibody? For example, you have a fluorochrome conjugated primary antibody that is raised in goat (the host) against X antigen of mouse, the Goat anti-mouse X antibody of IgG2a isotype. Two major reactions will occur inside the tube, which would result in fluorescence staining. They are as follows; 1. The primary antibody will recognize the X antigen specifically and will result in positive staining or fluorescence 2. The FC region of your primary antibody that belongs to the host species where it has been raised, in this case, the Goat, will bind non-specifically to the FC receptors present in the mouse cells (B cells, macrophages, etc.,). This will result in non-specific or background fluorescence. In addition, the Goat originated constant region may produce other non-specific protein interactions, which will also result in background florescence. Therefore, to deduct this non-specific or background staining, you have to asses the level of back ground staining. For this purpose, you have to use a flurochrome conjugated non-specific antibody, which is consisted of FC region of the host species,with the same isotype, in this case, the goat immunoglobulin, IgG2a (Goat IgG2a). This is called isotype control antibody. The level of fluorescence resulting from staining with the isotype control will be the non-specific binding or the background fluorescence. So, basically your Isotype control should be matched with the host species of your primary antibody and the isotype. It should be conjugated to the same fluorochrome as the primary antibody and you should use the same concentration as the primary antibody in the staining. To get rid of FC mediated background staining you can use an appropriate FC receptor blocking antibody prior to the primary antibody. Hope I addressed your question.

you do not have much choice with enzyme immuno reaction, it is going to be diffucult to do three color using it, since peroxidase has two substrates which can be used for development of red (AEC), brown (DAB), black (DAB+Co), brownish green (DAB+Ni). ALP will give a red color with its substrates. you cannot use two HRP antibodies to get a separate reaction with AEC and DAB. so the only choice is IF.

thanks a lot to everybody for discussion. And I will start do some experiment according your advice. Taras, I may need your help if I decide to do immunolocalization.

ok . very thanks for your scientific response . but I have question : is it possible , during make asthma by ova TLR-4 mRNA upregulate in PBMC or airway epithelial cell ?

While I have no direct answer for your specific question, I would warn you that all T cells are not equal in their responses and all antibodies are not equal either! J Exp Med. 1984 Aug 1;160(2):494-505. T cell antiidiotypic antibodies reveal differences between two human leukemias. Posnett DN, Bigler RD, Bushkin Y, Fisher DE, Wang CY, Mayer LF, Chiorazzi N, Kunkel HG.

Thanks to you all for your answers and your help. I use infected DCs @24h post-infection. I agree that for moDCs irradiation is not useful because they do not proliferate, my doubt was rather for BMDCs.

Jingyun, I didn't realize that the specificity of your mAb (IgG2a) was also changed in addition to the isotype switch. It seems to me that a reasonable explanation is the accidental infection of the mice used to expand the hybridoma. Your choice here is to try to replicate this process, but with a virus, preferable one infecting DC. If I understand correctly, your ligand for the IgG2a is a DC surface protein. Some strains of LCMV infect preferentially DC, but would be safer to use inactivated virus. LCMV can quickly spread in a mouse colony if not properly handled. Have you considered an alternative like to isolate DC membranes to start a fresh immunization protocol?