Hi,
I am having a hard time replicating published in vitro Fn proliferation experiments with HCT116 cells in the following two papers;
1) Fusobacterium nucleatum Promotes Chemoresistance to Colorectal Cancer by Modulating Autophagy
July 2017 Cell 170(3):548-563.e16 DOI: 10.1016/j.cell.2017.07.008
2) Fusobacterium nucleatum Increases Proliferation of Colorectal Cancer Cells and Tumor Development in Mice by Activating TLR4 Signaling to NFκB, Upregulating Expression of microRNA-21
November 2016 Gastroenterology 152(4) DOI: 10.1053/j.gastro.2016.11.018
Briefly, I am plating ~50K cells per well in 24 well plate with DMEM suppl. 10% FBS (without Pen/Strep) and adding log phase bacteria with increasing MOIs (0 MOI, 100 MOI, 500 MOI and 1000 MOI). The bacteria is also resuspended in the same growth medium (again, without Pen/Strep).
I tried both ATCC25586 and EAVG_002 strains.
I incubate the Fn-HCT116 plates in regular mammalian cell culture incubator (37C and 5% CO2) and measure cell viability/count on Day-1, Day-2 and Day-3 using Trypan Blue exclusion method.
I am seeing cell death in a both dose-dependent and time-dependent manners. That is, the HCT116 cells are dying over time and cell death is increasingly higher with increased MOI.
does anyone have any suggestions as to what I might be missing etc.?
thank you in advance!
OZ
"CRC cells were seeded in 24-well plates at 1 × 104 cells per well in the growth medium. Cells were untreated or incubated with FadAc or peptides at the indicated concentration or with bacteria at an moi of 1000:1. Cell numbers were counted at 24 hr intervals using a hemocytometer. Each experiment was performed in triplicate and repeated at least twice." This is directly from Rubistein 2013 Cell paper, have you try using less cells?, you mention you used 50K cells for a P24. Can you comment if you resolved your issues :) Regards Jenny
Thanks Jennifer Alfaro yes I tried different cell numbers per well, in both 6 well plate and 24 well plate. All led to death of the cells starting from 6h post co-incubation.
I also tried resuspending the bacteria in sterile DPBS and/or cell culture growth medium prior to adding into the wells that have HCT116 or HT29 cells.
In addition, I tried to add the bacteria before and after the CRC cell lines are adhered to the plate.
The problem still persists. I am not sure how to get around that.
do you have any suggestion if I am missing a point?
thanks a lot,
best!
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