I would like to deplet CD4 and CD8 T cells in vivo. But I do not have a flow cytometer which I could use to check if the in vivo depletion was successful. So, could someone tell which other method I could use to check if I succeeded in depleting these cells in vivo using clone GK1.5 and 53-6.7 antibodies?
Determination of percentages of CD4+ and CD8+ T cells can be done using ELISA assay. Please see the attached paper with protocol. But you will need plate reader for the ELISA
Hi Jerome,
alternative methods could be 1) an immunofluorescent microscopy or 2) a CD4/CD8 specific isolation and counting.
1) For microscopy, you will need of course corresponding microscope: you have to fix and stain your cells in a plate with mentioned primary antibodies (GK1.5 and 53-6.7), and on top of them use conjugated anti- (mouse, rat... depends on the origin of your primary Ab) antibodies. Conjugated fluorochrome should fit microscope settings. Just apply the protocol for T cells immunostaining for microscopy.
2) The second way is to use CD3 or CD4 or CD8 specific beads to isolate cells from the sample and then simple count cells numbers in a positive or negative fraction. For negative fraction - if in vivo depletion was successful then beads not reduce cell numbers significantly (compared to the undepleted control where you will see significant cell number reduction) or if you use magnetic beads... you can apply CD3 isolation protocol (MACS Miltenyi protocol for cells isolation) and directly see how many positive cells you can obtain from the sample compare to control and make a conclusion how effective (%) was in vivo depletion = (absolute cell number from depleted sample / abs. cell number from control) x 100. However magnetic beads are quite expensive to use them as a tool for specific cells counting. But it is easy to use and you don't need any expensive equipment, only a light microscope. Will take maximum 50 min.
Good luck!
The most economical and accessible answer is the use of ELISA, repeat each sample in triplicate and be sure to process in addition to the controls of commercial kits, another sample of concentration known as additional control to your working protocol.
Hi dear, i think two alternative method you can use, 1 ) flourecent micoscope or
2) magnetic bead. Magnetic bead is so easy to use
Thank you all for your suggestions. They were really helpful !!
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