T cell activation via CD3 and/or Cd28 need to solve a problem - T cell activation cd3 (Feb/27/2007 )
Hi everyone,
I am working very hard to get phosphorylation signal after T cell activation. First, I thought that my antibody(WB) did'nt work well, but finally, I think that the problem is at the stimulation step. To verify that hypothesis, I used these antibodies (anti-CD3 5 ug/ml clone: ucht1 and co-stimulatory signal anti CD-28 5 ug/ml, clone 28.2). These antibodies co-incubated (but not fixed to the plate, does that change something? I don't think it will, I need to incubate very short time in eppendorfs for WB stimulation) in a 96 wells plate with T lymphocytes at 3 different concentrations, but the result after 3 and 4 days in the incubator is exactly the same than non-stimulated cells (using a MTT method), so there is no cell proliferation.
When I use the same stimulation to do my western blot, I use the same both antibodies (same concentration) incubated with about 5 000 000 T lymphocytes on ice for 20 minutes then after I put them in a 37 degree water bath for different time (0, .5 min, 1 min, 2 min, 5 min, 10 min and 30 min). even I did an IP to concentrate the proteins, but no results. Sure I use a protease and a phosphatase inhibitor cocktail. I really don't know where the problem can come except from the stimulation.
Could someone can help me?
Thank you for your help!
Have a great day!
Brassap
-brassap-
QUOTE (brassap @ Feb 27 2007, 12:17 PM)
Hi everyone,
I am working very hard to get phosphorylation signal after T cell activation. First, I thought that my antibody(WB) did'nt work well, but finally, I think that the problem is at the stimulation step. To verify that hypothesis, I used these antibodies (anti-CD3 5 ug/ml clone: ucht1 and co-stimulatory signal anti CD-28 5 ug/ml, clone 28.2). These antibodies co-incubated (but not fixed to the plate, does that change something? I don't think it will, I need to incubate very short time in eppendorfs for WB stimulation) in a 96 wells plate with T lymphocytes at 3 different concentrations, but the result after 3 and 4 days in the incubator is exactly the same than non-stimulated cells (using a MTT method), so there is no cell proliferation.
When I use the same stimulation to do my western blot, I use the same both antibodies (same concentration) incubated with about 5 000 000 T lymphocytes on ice for 20 minutes then after I put them in a 37 degree water bath for different time (0, .5 min, 1 min, 2 min, 5 min, 10 min and 30 min). even I did an IP to concentrate the proteins, but no results. Sure I use a protease and a phosphatase inhibitor cocktail. I really don't know where the problem can come except from the stimulation.
Could someone can help me?
Thank you for your help!
Have a great day!
Brassap
I am working very hard to get phosphorylation signal after T cell activation. First, I thought that my antibody(WB) did'nt work well, but finally, I think that the problem is at the stimulation step. To verify that hypothesis, I used these antibodies (anti-CD3 5 ug/ml clone: ucht1 and co-stimulatory signal anti CD-28 5 ug/ml, clone 28.2). These antibodies co-incubated (but not fixed to the plate, does that change something? I don't think it will, I need to incubate very short time in eppendorfs for WB stimulation) in a 96 wells plate with T lymphocytes at 3 different concentrations, but the result after 3 and 4 days in the incubator is exactly the same than non-stimulated cells (using a MTT method), so there is no cell proliferation.
When I use the same stimulation to do my western blot, I use the same both antibodies (same concentration) incubated with about 5 000 000 T lymphocytes on ice for 20 minutes then after I put them in a 37 degree water bath for different time (0, .5 min, 1 min, 2 min, 5 min, 10 min and 30 min). even I did an IP to concentrate the proteins, but no results. Sure I use a protease and a phosphatase inhibitor cocktail. I really don't know where the problem can come except from the stimulation.
Could someone can help me?
Thank you for your help!
Have a great day!
Brassap
yes, whether they are complexed to the plate or not does matter. soluble CD3 is more of an inhibitory signal while plate bound is stimulatory. Also, we use twice the concentration of CD28 that you are using.
-tlblase-
QUOTE (tlblase @ Feb 27 2007, 03:41 PM)
QUOTE (brassap @ Feb 27 2007, 12:17 PM)
Hi everyone,
I am working very hard to get phosphorylation signal after T cell activation. First, I thought that my antibody(WB) did'nt work well, but finally, I think that the problem is at the stimulation step. To verify that hypothesis, I used these antibodies (anti-CD3 5 ug/ml clone: ucht1 and co-stimulatory signal anti CD-28 5 ug/ml, clone 28.2). These antibodies co-incubated (but not fixed to the plate, does that change something? I don't think it will, I need to incubate very short time in eppendorfs for WB stimulation) in a 96 wells plate with T lymphocytes at 3 different concentrations, but the result after 3 and 4 days in the incubator is exactly the same than non-stimulated cells (using a MTT method), so there is no cell proliferation.
When I use the same stimulation to do my western blot, I use the same both antibodies (same concentration) incubated with about 5 000 000 T lymphocytes on ice for 20 minutes then after I put them in a 37 degree water bath for different time (0, .5 min, 1 min, 2 min, 5 min, 10 min and 30 min). even I did an IP to concentrate the proteins, but no results. Sure I use a protease and a phosphatase inhibitor cocktail. I really don't know where the problem can come except from the stimulation.
Could someone can help me?
Thank you for your help!
Have a great day!
Brassap
I am working very hard to get phosphorylation signal after T cell activation. First, I thought that my antibody(WB) did'nt work well, but finally, I think that the problem is at the stimulation step. To verify that hypothesis, I used these antibodies (anti-CD3 5 ug/ml clone: ucht1 and co-stimulatory signal anti CD-28 5 ug/ml, clone 28.2). These antibodies co-incubated (but not fixed to the plate, does that change something? I don't think it will, I need to incubate very short time in eppendorfs for WB stimulation) in a 96 wells plate with T lymphocytes at 3 different concentrations, but the result after 3 and 4 days in the incubator is exactly the same than non-stimulated cells (using a MTT method), so there is no cell proliferation.
When I use the same stimulation to do my western blot, I use the same both antibodies (same concentration) incubated with about 5 000 000 T lymphocytes on ice for 20 minutes then after I put them in a 37 degree water bath for different time (0, .5 min, 1 min, 2 min, 5 min, 10 min and 30 min). even I did an IP to concentrate the proteins, but no results. Sure I use a protease and a phosphatase inhibitor cocktail. I really don't know where the problem can come except from the stimulation.
Could someone can help me?
Thank you for your help!
Have a great day!
Brassap
yes, whether they are complexed to the plate or not does matter. soluble CD3 is more of an inhibitory signal while plate bound is stimulatory. Also, we use twice the concentration of CD28 that you are using.
Thank you very much for that fast and usefull answer. So if I understand, for cell proliferation assay, I should fix the antibodies (CD3 and CD28) to the plate (in the activation wells) and then I will be able to measure the proliferation. Should I use carbonate buffer to fix the antibodies to the plate? Do you have a protocol to suggest to me?
The other problem is that, many articles are activating T lymphocytes in small tubes with CD3/CD28 antibodies, how they can get increase in phosphorylation if they use the CD3 in a soluble form.
I really thank you for your help, once I solved these two problems, my life will change
Brassap
-brassap-
QUOTE (brassap @ Feb 27 2007, 01:18 PM)
QUOTE (tlblase @ Feb 27 2007, 03:41 PM)
QUOTE (brassap @ Feb 27 2007, 12:17 PM)
Hi everyone,
I am working very hard to get phosphorylation signal after T cell activation. First, I thought that my antibody(WB) did'nt work well, but finally, I think that the problem is at the stimulation step. To verify that hypothesis, I used these antibodies (anti-CD3 5 ug/ml clone: ucht1 and co-stimulatory signal anti CD-28 5 ug/ml, clone 28.2). These antibodies co-incubated (but not fixed to the plate, does that change something? I don't think it will, I need to incubate very short time in eppendorfs for WB stimulation) in a 96 wells plate with T lymphocytes at 3 different concentrations, but the result after 3 and 4 days in the incubator is exactly the same than non-stimulated cells (using a MTT method), so there is no cell proliferation.
When I use the same stimulation to do my western blot, I use the same both antibodies (same concentration) incubated with about 5 000 000 T lymphocytes on ice for 20 minutes then after I put them in a 37 degree water bath for different time (0, .5 min, 1 min, 2 min, 5 min, 10 min and 30 min). even I did an IP to concentrate the proteins, but no results. Sure I use a protease and a phosphatase inhibitor cocktail. I really don't know where the problem can come except from the stimulation.
Could someone can help me?
Thank you for your help!
Have a great day!
Brassap
I am working very hard to get phosphorylation signal after T cell activation. First, I thought that my antibody(WB) did'nt work well, but finally, I think that the problem is at the stimulation step. To verify that hypothesis, I used these antibodies (anti-CD3 5 ug/ml clone: ucht1 and co-stimulatory signal anti CD-28 5 ug/ml, clone 28.2). These antibodies co-incubated (but not fixed to the plate, does that change something? I don't think it will, I need to incubate very short time in eppendorfs for WB stimulation) in a 96 wells plate with T lymphocytes at 3 different concentrations, but the result after 3 and 4 days in the incubator is exactly the same than non-stimulated cells (using a MTT method), so there is no cell proliferation.
When I use the same stimulation to do my western blot, I use the same both antibodies (same concentration) incubated with about 5 000 000 T lymphocytes on ice for 20 minutes then after I put them in a 37 degree water bath for different time (0, .5 min, 1 min, 2 min, 5 min, 10 min and 30 min). even I did an IP to concentrate the proteins, but no results. Sure I use a protease and a phosphatase inhibitor cocktail. I really don't know where the problem can come except from the stimulation.
Could someone can help me?
Thank you for your help!
Have a great day!
Brassap
yes, whether they are complexed to the plate or not does matter. soluble CD3 is more of an inhibitory signal while plate bound is stimulatory. Also, we use twice the concentration of CD28 that you are using.
Thank you very much for that fast and usefull answer. So if I understand, for cell proliferation assay, I should fix the antibodies (CD3 and CD28) to the plate (in the activation wells) and then I will be able to measure the proliferation. Should I use carbonate buffer to fix the antibodies to the plate? Do you have a protocol to suggest to me?
The other problem is that, many articles are activating T lymphocytes in small tubes with CD3/CD28 antibodies, how they can get increase in phosphorylation if they use the CD3 in a soluble form.
I really thank you for your help, once I solved these two problems, my life will change
Brassap
I can't tell you about the tubes but as for our protocal we immobilize aCD3 by simply putting it in PBS without Ca/Mg for at least 2 hrs at RT although some people do also use a coating buffer (i did in my previous lab) so the latter is a preference thing. The aCD28 does not need to be immobliized. You should see proliferation as soon as day 3 although with human cells we look at day 4 or 5. Good luck
-tlblase-
I use ConA at 1ug/mL to activate without antigen present, or coat 96wells with 100uL of 2ug/mL anti-CD3 in PBS for 2 hrs before adding cells.
-bwhhms-
QUOTE (bwhhms @ Feb 28 2007, 04:39 PM)
I use ConA at 1ug/mL to activate without antigen present, or coat 96wells with 100uL of 2ug/mL anti-CD3 in PBS for 2 hrs before adding cells.
Con A sure is a good idea, but the problem is that I have to continue studies of a guy who left and I try to do what he did (co-stimulation CD3-CD28 in eppendorfs for few seconds to few minutes) but I don't have phosphorylation signals and I use all the possible phosphatases inhibitors.
Thank you very much for your help and your fast answer!
Have a good day!
Brassap
-brassap-