am I right or not about this gating from Nilotpal Banerjee's blog

we are going to distinguish NKcell population within PBMC through flow cytometry. Our approach is as follows:- 1. From the dot plot of FSC vs SSC we r gating CD-3 Cy5|| CD-56 FITC. 2. To isolate NK cell We are choosing the CD3-/CD56+ cells. 3. As we wish to detect the NK cell dim/bright population, we then gate CD56 FITC||CD16PE population. 4.At this stage we have isolated NK population and also differentiated NK dim/bright cells also. 5. Next we are going to observe CD-328 PE along with CD56 FITC dim/bright. 6. Similarly CD-329 PE will be observed. IMPORTANT NOTE: Our constraints:- 1.we can not perform 6 color facs. 2. We have isotype control having 3 fluorophores namely FITC, PE, Cy5 which can't be altered due to budget constraint. 3.we have access to a BDFacscalibur having 4color only namely FL1,FL2,FL3,FL4. We prefer to perform 2color face. 4. Due to budget constraint , we will set the CD3 and CD16 gates first and then further just gate the PBMC through the previously selected gate. Then we will just tag the PBMC with CD56 & CD328/CD329 is it okay? Waiting for your kind reply.

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By Nilotpal Banerjee
Added Jun 3 '15

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