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RAW264.7 cells+nitric oxide determination - (Jul/26/2009 )

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Hello,
I am trying to stimulate RAW cells with LPS and Pam3Cys4K for Nitric oxide determination using griess assay. The standard curves I get is nice and linear but my doubt is
1. The OD values in the empty wells or or in the 0uM nitrite standard well reads as 0.05.
Therefore should I minus this base value from the OD value obtained for the samples and then compare with the nitrite standard curve for measuring nitrite concentration?. If i do it this way then there seems to be no Nitrite production at all, for example on day day 4 I get a OD value of0.11 which corresponds to about 25uM of Nitrite according to the standard curve. But the OD value in the 0uM level in the nitrite standard curve is 0.05. So should I minus this value from 0.11 which would then be 0.06 and then look at the corresponding nitrite level from the standard curve. Hope You can understand what I am trying to say. Kindly help! -Shy

-shyarasu-

Either subtract 0.05 off everything, including the standard curve, thus making the std curve go through 0,0; or donít subtract anything (unless you have a media blank as well) in which case the std curve will have an intercept of 0.05. You should get the same answer both ways.

-DRT-

shyarasu on Jul 27 2009, 05:58 AM said:

Hello,
I am trying to stimulate RAW cells with LPS and Pam3Cys4K for Nitric oxide determination using griess assay. The standard curves I get is nice and linear but my doubt is
1. The OD values in the empty wells or or in the 0uM nitrite standard well reads as 0.05.
Therefore should I minus this base value from the OD value obtained for the samples and then compare with the nitrite standard curve for measuring nitrite concentration?. If i do it this way then there seems to be no Nitrite production at all, for example on day day 4 I get a OD value of0.11 which corresponds to about 25uM of Nitrite according to the standard curve. But the OD value in the 0uM level in the nitrite standard curve is 0.05. So should I minus this value from 0.11 which would then be 0.06 and then look at the corresponding nitrite level from the standard curve. Hope You can understand what I am trying to say. Kindly help! -Shy



The detection limit for that assay is 3uM...anything less than that is unreliable. RAW cells can produce huge quantities of NO2-, upto 30-40uM in a 24 hour experiment.

The LPS used is very important and the amount of NO2- produced is dependent upon the source of LPS. In our hands LPS sourced from WS Typhosa gives greater levels of NO2- than LPS derived from E.coli.

The number of cells in your well is also important. If yuo have too few cells then again it will be difficult to measure NO2-.

Your 0uM concentration on your standard curve should not give you much of an OD reading at all. Check what media you are using...this is also important as DMEM has no Nitrate/Nitrite...whereas RPMI has mM amounts of Nitrate. Always subtract the 0uM value from all the other OD...because this is your true baseline reading

Hope this is useful

Kindest regards

Rhombus

-rhombus-

rhombus on Aug 10 2009, 09:32 PM said:

The LPS used is very important and the amount of NO2- produced is dependent upon the source of LPS. In our hands LPS sourced from WS Typhosa gives greater levels of NO2- than LPS derived from E.coli.


Cheers rhombus, thatís a handy tip. Does changing LPS also change the way RAWs respond to anti-inflammatory compounds? I would assume not if the compounds are only affecting the synthase.

-DRT-

DRT on Aug 10 2009, 04:44 PM said:

rhombus on Aug 10 2009, 09:32 PM said:

The LPS used is very important and the amount of NO2- produced is dependent upon the source of LPS. In our hands LPS sourced from WS Typhosa gives greater levels of NO2- than LPS derived from E.coli.


Cheers rhombus, thatís a handy tip. Does changing LPS also change the way RAWs respond to anti-inflammatory compounds? I would assume not if the compounds are only affecting the synthase.

Thanks a lot for that! I have one more doubt..
I would like to know till which passage I can use the RAW 264.7 cells. I am currently at P: 22 and I find that the viability is less compared to earlier ones and also the level of nitric oxide produced also seems to be falling. Please help!

-shyarasu-

shyarasu on Sep 1 2009, 07:56 AM said:

DRT on Aug 10 2009, 04:44 PM said:

rhombus on Aug 10 2009, 09:32 PM said:

The LPS used is very important and the amount of NO2- produced is dependent upon the source of LPS. In our hands LPS sourced from WS Typhosa gives greater levels of NO2- than LPS derived from E.coli.


Cheers rhombus, thatís a handy tip. Does changing LPS also change the way RAWs respond to anti-inflammatory compounds? I would assume not if the compounds are only affecting the synthase.

Thanks a lot for that! I have one more doubt..
I would like to know till which passage I can use the RAW 264.7 cells. I am currently at P: 22 and I find that the viability is less compared to earlier ones and also the level of nitric oxide produced also seems to be falling. Please help!



Dear shyarasu,

It all depends upon how you grow your cells. I assume that you grow them in plastic TC flasks. RAWs will stick like superglue to the TC plastic and scraping is usually the method of choice...because trypsin will not toucjh them. Consequently the cells with be damaged each time you passage them and the viability will reduce over time.

If you grow them as they should be i.e in suspension....then there is no trypsinisation or scraping and the cells will stay viable for longer.

In my experience RAW's and J774's can be passaged upto 200 times...without loss of viability or loss in their ability to induce iNOS....if you grow them in SUSPENSION.

Hope this is useful

Kindest regards

Uncle Rhombus

-rhombus-

rhombus on Sep 2 2009, 09:11 PM said:

shyarasu on Sep 1 2009, 07:56 AM said:

DRT on Aug 10 2009, 04:44 PM said:

rhombus on Aug 10 2009, 09:32 PM said:

The LPS used is very important and the amount of NO2- produced is dependent upon the source of LPS. In our hands LPS sourced from WS Typhosa gives greater levels of NO2- than LPS derived from E.coli.


Cheers rhombus, thatís a handy tip. Does changing LPS also change the way RAWs respond to anti-inflammatory compounds? I would assume not if the compounds are only affecting the synthase.

Thanks a lot for that! I have one more doubt..
I would like to know till which passage I can use the RAW 264.7 cells. I am currently at P: 22 and I find that the viability is less compared to earlier ones and also the level of nitric oxide produced also seems to be falling. Please help!



Dear shyarasu,

It all depends upon how you grow your cells. I assume that you grow them in plastic TC flasks. RAWs will stick like superglue to the TC plastic and scraping is usually the method of choice...because trypsin will not toucjh them. Consequently the cells with be damaged each time you passage them and the viability will reduce over time.

If you grow them as they should be i.e in suspension....then there is no trypsinisation or scraping and the cells will stay viable for longer.

In my experience RAW's and J774's can be passaged upto 200 times...without loss of viability or loss in their ability to induce iNOS....if you grow them in SUSPENSION.

Hope this is useful

Kindest regards

Uncle Rhombus


Dear Rhombus

Hi, Now that I'm about to start to culture Raw cell, and gonna try to do suspension cell culture as you said..
but There IS NO Teche stirer bottle in our lab.. Is there any other way to do suspension culture?.. Would it be possible if I culture them in non coated petri-dish? Actually I tried that yesterday and Weired things happened.. half of them were slightly attached and otherws were floated with cluster..
Hope to see your answer

-Sumyung-

Hello all,
Have any of you worked with synthetic lipopeptide (Pam3Cys4K)?. I am doing the Griess assay with Pam3 as my positive control. It was working fine until we started this new cell line. And now the LPS is still giving results but the Pam3 for some unknown reason does not produce NO or even if produced quantity very low. Do any of you have an idea as to why this is happening and how I can fix this? Thank you
Shyarasu
Attached File

-shyarasu-

rhombus on Mon Aug 10 11:32:03 2009 said:


shyarasu on Jul 27 2009, 05:58 AM said:

Hello,
I am trying to stimulate RAW cells with LPS and Pam3Cys4K for Nitric oxide determination using griess assay. The standard curves I get is nice and linear but my doubt is
1. The OD values in the empty wells or or in the 0uM nitrite standard well reads as 0.05.
Therefore should I minus this base value from the OD value obtained for the samples and then compare with the nitrite standard curve for measuring nitrite concentration?. If i do it this way then there seems to be no Nitrite production at all, for example on day day 4 I get a OD value of0.11 which corresponds to about 25uM of Nitrite according to the standard curve. But the OD value in the 0uM level in the nitrite standard curve is 0.05. So should I minus this value from 0.11 which would then be 0.06 and then look at the corresponding nitrite level from the standard curve. Hope You can understand what I am trying to say. Kindly help! -Shy



The detection limit for that assay is 3uM...anything less than that is unreliable. RAW cells can produce huge quantities of NO2-, upto 30-40uM in a 24 hour experiment.

The LPS used is very important and the amount of NO2- produced is dependent upon the source of LPS. In our hands LPS sourced from WS Typhosa gives greater levels of NO2- than LPS derived from E.coli.

The number of cells in your well is also important. If yuo have too few cells then again it will be difficult to measure NO2-.

Your 0uM concentration on your standard curve should not give you much of an OD reading at all. Check what media you are using...this is also important as DMEM has no Nitrate/Nitrite...whereas RPMI has mM amounts of Nitrate. Always subtract the 0uM value from all the other OD...because this is your true baseline reading

Hope this is useful

Kindest regards

Rhombus

Hi,
I am culturing J774 cells in DMEM.During experiment i treated cells with 1microgram/ml LPS for 20 hours.
From Griess assay ,OD reading in blank (just media-DMEM) was 0.26 and LPS treated ones is 0.29.
For me, the optical density reading from Blank and LPS treated cells are both looking same.I am doing experiment for first time and feel struck.
Can anyone help me with this.-Thanks... Nethra

-nethra-

nethra on Tue Aug 16 22:58:01 2011 said:


rhombus on Mon Aug 10 11:32:03 2009 said:


shyarasu on Jul 27 2009, 05:58 AM said:

Hello,
I am trying to stimulate RAW cells with LPS and Pam3Cys4K for Nitric oxide determination using griess assay. The standard curves I get is nice and linear but my doubt is
1. The OD values in the empty wells or or in the 0uM nitrite standard well reads as 0.05.
Therefore should I minus this base value from the OD value obtained for the samples and then compare with the nitrite standard curve for measuring nitrite concentration?. If i do it this way then there seems to be no Nitrite production at all, for example on day day 4 I get a OD value of0.11 which corresponds to about 25uM of Nitrite according to the standard curve. But the OD value in the 0uM level in the nitrite standard curve is 0.05. So should I minus this value from 0.11 which would then be 0.06 and then look at the corresponding nitrite level from the standard curve. Hope You can understand what I am trying to say. Kindly help! -Shy



The detection limit for that assay is 3uM...anything less than that is unreliable. RAW cells can produce huge quantities of NO2-, upto 30-40uM in a 24 hour experiment.

The LPS used is very important and the amount of NO2- produced is dependent upon the source of LPS. In our hands LPS sourced from WS Typhosa gives greater levels of NO2- than LPS derived from E.coli.

The number of cells in your well is also important. If yuo have too few cells then again it will be difficult to measure NO2-.

Your 0uM concentration on your standard curve should not give you much of an OD reading at all. Check what media you are using...this is also important as DMEM has no Nitrate/Nitrite...whereas RPMI has mM amounts of Nitrate. Always subtract the 0uM value from all the other OD...because this is your true baseline reading

Hope this is useful

Kindest regards

Rhombus

Hi,
I am culturing J774 cells in DMEM.During experiment i treated cells with 1microgram/ml LPS for 20 hours.
From Griess assay ,OD reading in blank (just media-DMEM) was 0.26 and LPS treated ones is 0.29.
For me, the optical density reading from Blank and LPS treated cells are both looking same.I am doing experiment for first time and feel struck.
Can anyone help me with this.-Thanks... Nethra



Dear Nethra,

The Griess is an colour end point assay. As stated previously, the assay lower limit is around 2-3uM. Your standard curve should be:
0uM
3Um
10uM
30uM
100uM
300uM

548nM is the wavelength you need to read the absorbance. What is your standard curve like????

It seems obvious from your results that the cells have not produced Nitric oxide ( Nitrite).

Some things to check:

Try different sources of LPS....we use W.S. Typhosa for our inductions. I prepare the LPS fresh every time and use 10ug/ml which gives approximately 80% production of NO. We havew in the past tried to use LPS from E.coli....which in my hands give a very weak induction.

Once you have identified which source of LPS......do a dose response for the LPS. I use J774 from passage 20 to passage 200. They give exactly the same induction profile over all those passages.......IF YOU LOOK AFTER THE CELLS.

Have you checked the viability of the cells you are using?????

Do you regularly check for levels of Endotoxin in media and serum.....THIS IS VERY IMPORTANT....as very low levels of background endotoxin will inhibit normal LPS induction of iNOS.

Under our conditions i.e. a 6 well plate with 500,000 cells over a 20 hour time point, stimulated with 10ug/ml of LPS (W.S.Typhosa) will give between 25 and 30uM Nitrite values when measured by the Griess reaction.

I hope this proves useful.

My kindest regards.

Uncle Rhombus.

-rhombus-
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