Hi, all, I need help for protocols of shRNA knockdown effect on Ag-specific Treg differentiation in vitro and/or in vivo.
Now, I want to check the effect of my shRNA on Ag-specific Treg differentiation both in vitro and in vivo. But I could not find the proper protocols.
1. For in vitro assay, I thought I should first stimulate naive OT-II T cells with OVAp and irradiated APC, then do shRNA included retrovirus infection 2 days after T cell stimulation. But I was told by several persons that this procedure would not work because virus can not reach T cells since APC is there. So it means I have to use anti-CD3 and anti-CD28 instead of APC? I am not sure about it. And I wonder if I can overcome this problem by using puromycin selection after virus infection or not. I need suggestions from experienced experts and thanks a lot in advance!
2. For in vivo assay, I plan to use puromycin selection after retrovirus infection. I just wonder usually after shRNA included retrovirus infection how long I should culture the OT-II T cells with puromycin before doing adoptive transfer. I cannot find enough such information from publications.
If somebody can share his protocols or give any suggestions, I appreciate it very much!
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#89245 Help: protocols for shRNA knockdown effect on Ag-specific Treg differentiation
Posted by
on 11 October 2010 - 09:06 AM
in
siRNA, microRNA and RNAi
#89244 Help:need protocols for Ag-specific Treg differentiation in vitro and/or in vivo
Posted by
on 11 October 2010 - 08:52 AM
in
Immunology
Hi, all, I need help for protocols of Ag-specific Treg differentiation in vitro and/or in vivo.
Now, I want to check the effect of my shRNA on Ag-specific Treg differentiation both in vitro and in vivo. But I could not find the proper protocols.
1. For in vitro assay, I thought I should first stimulate naive OT-II T cells with OVAp and irradiated APC, then do shRNA included retrovirus infection 2 days after T cell stimulation. But I was told by several persons that this procedure would not work because virus can not reach T cells since APC is there. So it means I have to use anti-CD3 and anti-CD28 instead of APC? I am not sure about it. And I wonder if I can overcome this problem by using puromycin selection after virus infection or not. I need suggestions from experienced experts and thanks a lot in advance!
2. For in vivo assay, I plan to use puromycin selection after retrovirus infection. I just wonder usually after shRNA included retrovirus infection how long I should culture the OT-II T cells with puromycin before doing adoptive transfer. I cannot find enough such information from publications.
If somebody can share his protocols or give any suggestions, I appreciate it very much!
Now, I want to check the effect of my shRNA on Ag-specific Treg differentiation both in vitro and in vivo. But I could not find the proper protocols.
1. For in vitro assay, I thought I should first stimulate naive OT-II T cells with OVAp and irradiated APC, then do shRNA included retrovirus infection 2 days after T cell stimulation. But I was told by several persons that this procedure would not work because virus can not reach T cells since APC is there. So it means I have to use anti-CD3 and anti-CD28 instead of APC? I am not sure about it. And I wonder if I can overcome this problem by using puromycin selection after virus infection or not. I need suggestions from experienced experts and thanks a lot in advance!
2. For in vivo assay, I plan to use puromycin selection after retrovirus infection. I just wonder usually after shRNA included retrovirus infection how long I should culture the OT-II T cells with puromycin before doing adoptive transfer. I cannot find enough such information from publications.
If somebody can share his protocols or give any suggestions, I appreciate it very much!
