There are atleast three approaches u can follow. Ist if the protein of interest is extracellular u can used cell free extracts. if ur protein of interest is intracellular u can use cell lysates. We had used the same approach our work but to deduce pathogenicity of V.cholerae. U can use same technique also. The link is given below.

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0076200

The third approach is co-culture of bacteria with HEK cells.

This paper may help u

http://www.jimmunol.org/content/176/2/1228.full

Many thanks Priyabrata! That helps a lot! I am interested in anything that the bacteria may have as long it is able to active my receptor. Therefore, I want to try everything, cell free extracts and cell lysates as well as coculture if possible.

I will check those links.

Kind Regards,

Cristina

Hi Cristina,

We use live bacteria in LB to infect BMDMs. After 3 to 4 h of infection, gentamycin is added to check overgrowth. I hope this may help you.

Many thanks Rajendra! I will check that as well,

Kind Regards and many thanks once again for your information,

Cristina

I have used an approach similar to Rajendras, but removed cells from the LB and grown them for a while in RPMI or DMEM before adding them to cells (centrifuge the bacteria onto the cells to ensure a better contact) - allowed to remain in contact for 1h then added gentamycin to prevent overgrowth.There are components of LB (yeast extract etc ) which could potentially have a stimulatory effect. I do not know if your HEKs are transfected with plasmids eg TLR4 -MD2 etc, but you may well find that you get activation of genes associated with the innate immune system due to the LPS / endotoxin component of your probiotic bacteria. Probiotic bacteria are sometimes only "probiotic" in the context of an intact gut.Given to cells in isolation they can well have stimulatory effects that are not dissimilar to"pathogenic" bacteria. Likewise "commensals" are only "harmless" if they are in an intact gut. In a breached gut or in the blood stream they are pathogens.

Ian Teo

Dear Ian,

Many thanks for your reply. Actually, the particular assay that I want to do is the analysis of the increased calcium intracellular influx that is produced once my receptor (a g protein coupled receptor) is activated. This assay is carried out in a machine that allow the analysis of this Ca influx immediately after add the sample (in my case the bacteria). Therefore, the incubation time of the bacteria with the cells would be just few seconds. However, I think that, as you said, some components of the bacteria media may still interfere with my assay and therefore I was wondering if I could grow the bacteria in HBSS /HEPES buffer, which is the buffer I use in my assay???

Many thanks in advance,

Cristina

I think that HBSS + HEPES might be a bit short on nutrients. Grow them in RPMI, spin down, wash with HBSS + HEPES, spin and resuspend in the same buffer. What are you going to use as controls for your "probiotic" bacteria?

Ian

Hi Ian,

Many thanks for your reply. I will use as control the bacteria media (so, or MRS broth (that is the media where I am growing my bacteria) or the assay buffer if I can resuspend my bacteria fianlly in that buffer. As I said, is going to be only few seconds the analysis so it should be fine, I dont think bacteria will be affected that fast. Anyway, maybe it would be fine only analyse the effect of the supernatant and nonviable cell extract? Do you think alive bacteria would have any effect if their supernatant and cell extract wouldnt?

Many thanks,

Cristina

It is likely that live or inactivated organisms will trigger off PAMPs etc. How quickly I do not know, nor how quickly Ca levels are affected. If using organisms in welIs are you going to spin the co-cultures to ensure good bacteria -cell contact?

Are you looking specifically at the effect of probiotics and comparing them to other strains? I was thinking more in terms of what you were using as probiotics and control strains.

Ian

Christina, check out our protocol to stably introduce bacteria into human cell cultures for long periods of time (>24 h). Basically, we used an aqueous two-phase system that "entraps" the bacteria in a droplet within the culture media. Using this technique, we were able to pattern several differnt bacterial strains on human cell cultures with no impact on the epithelial viability if the strain was not harmful. As well, we studied the impact of P. aeruginosa (a pathogen) on human epithelial cells and showed that it caused a localized impact within the culture dish.

The citation is Dwidar M, Leung BM, Yaguchi T, Takayama S, Mitchell RJ (2013) "Patterning Bacterial Communities on Epithelial Cells." PLoS ONE 8(6): e67165. doi:10.1371/journal.pone.0067165

Hope this helps.

Many thanks Ian and Robert.

Ian, I won't use any control strain as this is a new study so there is not any paper regarding strains activating this receptor. As positive control I only can use the natural ligand of the receptor. Also, I cannot spin the co-culture for this particular assay as the Ca influx has to be analysed immediately after the inoculation of the bacteria on the cells. I may try that for further analysis in another kind of assays, so many thanks for the suggestion.

Robert, really interesting paper. I will try that method in future studies which may require longer incubation times. Many thanks for taking the time to help me.

I think my best option for now is carry out the Ca assay screening with the bacteria supernatant, their cell extract and the non-viable cells.

Kind Regards,

Cristina

Hi Cristina,

We (Attana AB) have developed a method based on QCM technology (quartz crytal microbalance) to study interactions between adherent cells and ligands in a continuous flow system. In your case you could have HEK cells grown on a chip and pass bacteria over them in a flow rate of 20 ul/min, mimicking the physiological levels, and can be done in any HBST +BSA. After removing bound bacteria from the cell surface you could continue with you Ca influx experiment on the same system.

Hi Christina,

You could add bacteria in the medium which you use for the cell cultivation but without serum.

To test the specificity of bacterial effect you may use the inhert latex beads (comparable with the size of your bacteria). The effect of

live bacteria you could also compared to that of heat-killed

bacteria at the same concentrations.

Usually we use at least three different concentration of bacteria in the same experiments.

The reffernce:"Staphylococcus aureus-induced G2/M phase transition delay in host epithelial cells increases bacterial infective efficiency.

Alekseeva L, Rault L, Almeida S, Legembre P, Edmond V, Azevedo V, Miyoshi A, Even S, Taieb F, Arlot-Bonnemains Y, Le Loir Y, Berkova N.

PLoS One. 2013 May 23;8(5):e63279. doi: 10.1371/journal.pone.0063279. Print 2013.

Good luck

Nadia Berkova

Many thanks for your anwers,

Diluka, I will have to look at that better. I am not sure if it suits with my experiment as the calcium is released immediately after the activation of the receptor and it last no longer than 80 s. So, what I usually do, is analyse the intracelullar Ca levels during 80 s after inoculation of my sample (in this case would be bacteria) on my cells. Therefore, I think that in that way you propose, if the alive bacteria has any effect on my receptor, it would increase the Ca levels but this Ca would be gone before remove bacteria from the cell surface and I wont be able to detect it when running my plate in the Ca assay.

Nadia, many thanks for that reference! Sorry if it is a stupid question but for me it is first time working with bacteria...what you mean when you said that I need to use inhert latex beads to test the specificity of my bacteria? it wouldn't be fine just test the bacteria media as negative control?

Thanks a millions for your help!!!

Cristina

Christina, Your question is not stupid at all, it is a pertnant question. Not everybody makes this kind of control. But iIf you will try to publish your results in a good journal, you may be asked to proove the specifity of the described effect. If you will use the media as a negative control, you will control the substances which are excreted by the bacteria outside into the medium.

You can not exclude the presence of the active substances on the surfacec of bacteria. Moreover it is possible that the ordinary simple spheres, similar to some bacteria geometric form, will have an effect. You need to study the specific effect related to your bacteria.

Latex beads are the control of the specifity of the effect.

Many thanks again Nadia! I didnt think about that, it makes totally sense. I will use that then!

Really appreciated all your help,

Kind Regards,

Cristina

Hi Christina,

The University of Groningen in the Netherlands has done some pilot studies in the development of media that can both tolerate and grow both bacteria and mammalian cells. The results of the pilot were used as the media in the "race for the surface" work done by Guruprakash Subbiahdoss. I would highly recommend looking at his work to find a good medium to work with.

The University of Groningen has a combination of microbiologists and cell biologists working in cooperation in a single department which facilitated the developmental process for an optimal media.

Hi Christina,

My PhD work is on the co-culture of bacteria and mammalian cells. I would suggest you to refer my publications. If you have any questions, feel free to drop me an email. Good Luck !

Dear Brandon and Guruprakash,

Thank you very much for your suggestions.

Brandon, it sounds really interesting that media. I will have to look at it.

Guruprakash I will check your publications for sure! I'll let you know if I have any question,

Kind Regards,

Cristina

Hi Cristina,

I have looked through most of the suggestions so far and they seem to be in line with what my lab does.

I'm not too sure of all the experiments you are planning, but I'd like to give some suggestions from what it sounds like you might do.

It seems like you may be planning many experiments including "invasion" or "gentamicin protection" assays (http://en.wikipedia.org/wiki/Gentamicin_protection_assay), and there should be protocols easily available.

To counteract the problem of LB interfering with the assay, we grow some of our bacteria in base RPMI (or whatever the cell's media is). If you are concerned that this may change the bacteria's secretion profile, you could consider growing the bacteria in your normal bacterial medium, then washing with the mammalian cell media prior to adding (although this may also stimulate the bacteria).

To check the non-cell components' effects, you may want to use a "trans-well" system (we found that even ultra-centrifugation might not be a perfect separation tool). This "should" keep the bacteria out and allow everything else to pass through.

You may also want to try to compare the effects at different multiplicities of infection (MOI's) using some way of counting cells (have a set of wells for counting plated at the same density as the experimental wells) and bacteria (spectrophotometry).

I hope this helps.

Quais

Dear Quais,

Many thanks for taking the time to answer my question.

I am planning to carry out different in vitro screenings. I am going to start with the study of a receptor modulation activity of different bacteria strains. This receptor is a GPCR so what I do is analyse the increased intracellular Ca that is produced upon activation of the receptor. This Ca influx doesnt last too long, it has to be analysed immediately after add the bacteria, so, finally, for this particular assay I wont do coculture of the bacteria with the HEK cells. However, I will try your suggestions for further analysis that may need this incubation of the alive bacteria with the cells.

Regarding the comparation at different concentrations, I would also like compare between different bacteria strains. It is hard get exactly the same optical density for all of them, I guess it doesnt have to be too exact? I am use to work with cell culture in which you only have to count the cells easily with the cell counter but with bacteria is more difficult be sure you are actually testing same concentration in different strains as OD may vary within them.

Many thanks again,

Cristina

Dear Cristina

I can suggest you to read some of my publications. You will find some informations on co-culture between bacteria (probiotic, commensal or pathogen) and Caco-2 cells.

this is the link to one of them:

http://www.ncbi.nlm.nih.gov/pubmed/23122647

I hope it could help you

Yours sincerely

Nathalie Connil

Hi Nathalie,

Many thanks!!! I will check your publications,

Regards,

Cristina

I can recommend you my publications, I was interested in the adherence of bacteria to animal epithelial cells primoceltures, I can recommend you for reading these my publications: 1, Štyriak, I., Gálfi, P. and Kmet', V.: Adherence of ruminal Streptococcus bovis and Lactobacillus strains to primary and secondary cultures of rumen epithelium. Acta Microbiologica Hungarica, 39, 1992, 323-325.

2, Štyriak, I., Gálfi, P. and Kmet', V.: The adherence of three Streptococcus bovis strains to cells of rumen epithelium primoculture under various conditions. Arch. Anim. Nutr., 46, 1994, 357-365.

Best regards, Igor

Hi Igor,

Many thanks for your reply. I will check them and see if it can fit with what I have to do,

Best Regards,

Cristina

Hi Cristina, I am glad that I could help you. You will see whether these my publications will be useful for you. If I can help you anywhere, you can contact me.

Best regards, Igor

Hi Cristina, I am glad that I could help you. You will see whether these my publications will be useful for you. If I can help you anywhere, you can contact me.

Best regards, Igor