Anyone familiar with macrophage stimulation? - (Jul/15/2009 )
I have been doing stimulation of RAW264.7 with E.coli-LPS recently. The target of my experiment is LPS-induced superoxide production. However, the cells were not responsive after 24h of stimulation. This contradicts to the reference I came across. I think the problem could be the LPS. I remember someone here mentioned using freshly dissolved LPS for stimulation. Anyone familiar with that? All my LPS are in aliquots, I don't have new vial of lyophilized powder.
-Nrelo-
Nrelo on Jul 15 2009, 08:55 AM said:
I have been doing stimulation of RAW264.7 with E.coli-LPS recently. The target of my experiment is LPS-induced superoxide production. However, the cells were not responsive after 24h of stimulation. This contradicts to the reference I came across. I think the problem could be the LPS. I remember someone here mentioned using freshly dissolved LPS for stimulation. Anyone familiar with that? All my LPS are in aliquots, I don't have new vial of lyophilized powder.
Dear Nrelo,
It was probably my post that you remembered. I have used both RAW 264.7 and J774.A1 cell lines when using LPS, in my case to induce Nitric Oxide Synthase (iNOS). I always use fresh LPS, weighed out on the day of the experiment. I have a couple of suggestions:-
i) As you say the LPS has gone off..... we keep ours at +4oC lyophilised. This is usually stable over many years (>5yrs).
ii) Is the LPS you used exactly the same as the published data? .....i.e. there are many strains of E.coli and the derived LPS will have different activities.
iii) Have you recently mycoplasma tested your cells.......i.e. are they unresponsive to LPS because they are already being stimulated?
Cells can be contaminated with mycoplasma and not affect growth or morphology BUT affect other cellular parameters.
iv) Do you use antibiotics in your cell culture.... we have found that very small quantities of LPS switch off the cells ability to induce iNOS. If you are using antibiotics, there could be a cryptic contamination in your culture.
v). Have you changed your FBS/FCS recently........this is important as all serum has background levels of Endotoxin (LPS).......again could switch off the cells ability to produce superoxide.
vi). Is your superoxide assay working i.e. do you have a positive control in your protocol?
The easiest thing to do is
BUY MORE LPS
SEND CELLS FOR MYCOPLASMA TESTING
GETY FRESH CELLS
Hope this is useful
Kindest regards
Rhombus
-rhombus-
rhombus on Jul 15 2009, 04:43 AM said:
Nrelo on Jul 15 2009, 08:55 AM said:
I have been doing stimulation of RAW264.7 with E.coli-LPS recently. The target of my experiment is LPS-induced superoxide production. However, the cells were not responsive after 24h of stimulation. This contradicts to the reference I came across. I think the problem could be the LPS. I remember someone here mentioned using freshly dissolved LPS for stimulation. Anyone familiar with that? All my LPS are in aliquots, I don't have new vial of lyophilized powder.
Dear Nrelo,
It was probably my post that you remembered. I have used both RAW 264.7 and J774.A1 cell lines when using LPS, in my case to induce Nitric Oxide Synthase (iNOS). I always use fresh LPS, weighed out on the day of the experiment. I have a couple of suggestions:-
i) As you say the LPS has gone off..... we keep ours at +4oC lyophilised. This is usually stable over many years (>5yrs).
ii) Is the LPS you used exactly the same as the published data? .....i.e. there are many strains of E.coli and the derived LPS will have different activities.
iii) Have you recently mycoplasma tested your cells.......i.e. are they unresponsive to LPS because they are already being stimulated?
Cells can be contaminated with mycoplasma and not affect growth or morphology BUT affect other cellular parameters.
iv) Do you use antibiotics in your cell culture.... we have found that very small quantities of LPS switch off the cells ability to induce iNOS. If you are using antibiotics, there could be a cryptic contamination in your culture.
v). Have you changed your FBS/FCS recently........this is important as all serum has background levels of Endotoxin (LPS).......again could switch off the cells ability to produce superoxide.
vi). Is your superoxide assay working i.e. do you have a positive control in your protocol?
The easiest thing to do is
BUY MORE LPS
SEND CELLS FOR MYCOPLASMA TESTING
GETY FRESH CELLS
Hope this is useful
Kindest regards
Rhombus
ii) yes, the same as what the reference used
iii) no, I haven't test the cells yet
iv) the medium doesn't contain antibiotics
v) no, the serum was from the same bottle. The unstimulated cells produced similar background level of superoixde.
vi) I had PMA as positive control and the cells responsed quite well
Thx for your information. I will first try to work with the mycoplasma issue.
-Nrelo-