We have B6;CBA-Tg(Pou5f1-EGFP) animal model from Jackson. I am trying to collect primordial germ cells from the different embryonic stages. Acc. to available literature, there are 25000 PGCs in 13.5dpc gonads which is followed by mitotic arrest. Using FACS aria we tried isolating EGFP positive cells from 14.5dpc , 15.5dpc and 16.5dpc. In 14.5 dpc, only 500 cells per gonad were collected and in 15.5 and 16.5 dpc hardly any positivity was seen.
Briefly i am describing my protocol. We collected 7-8 male gonads from specific embryonic stage, washed them twice with ice cold DPBS. We added 1x Trypsin-EDTA ( Trypsin - EDTA Solution 1X w/ 0.025% Trypsin and 0.01% EDTA in Dulbecco’s Phosphate Buffered Saline) and incubated it at 37degree for 15 mins. After the incubation is complete, we removed trypsin EDTA and replaced it with DPBS and single cell suspension was made using 1ml syringe. The suspension was passed through 70um cell strainer and cells were directly taken for FACS sorting.
If anyone has experience with these cells. Please guide me.
Hi Poonam, If you want to confirm your doubts, you can verify the comparison on immunostaining of Germ cells at all the stages, and compare the stained sections. I am including one paper that may or may not help you in this regard that how to do that immunostaining if you wish to take an extra step. Figure 3 is for your attention in this paper enclosed, good luck.
Best
Subhash C. Juneja Thank your for your response. I will do that. I would like to share my one observation. As the PGCs in these animals are expressing OCT4-EGFP, for observing these gonads under florescence microscope, the exposure required to visualise 15.5dpc or 16.5dpc is more as compared to 14.5dpc. Therefore, while isolating cells from these advanced stages, we increased the laser power but it didn't result in any change.
Poonam Mehta I totally agree with you that because of low OCT4 activity, isolating germ cells from E15.5 and beyond gives low output. In this regard, I'll suggest going for trypsin incubation followed by mechanical shearing of the gonad and straining it utilizing the bigger cell size property of germ cell. There are several papers available in literature. PFA the literature below for FACS based sorting, maybe this can help you with GFP tagged germ cell isolation
Good Luck
Article Fluorescence Activated Cell Sorting of Live Female Germ Cell...
Poonam Mehta Use trypsin alone, exclude EDTA. Second set GFP and size-based gating for earlier stages where you can get a good amount of sorted cells output and replicate the same settings for sorting at advanced stages. To cross verify the purity go for immunostaining on cell smear using GC marker as suggested by Subhash C. Juneja and/or go for qPCR using GC and soma specific primers once. For any further queries DM me or Deepak N Modi.
I am highly impressed by Neha and Poonam and got the feel that, in India, high quality of research is being conducted. Congratulations.
Neha Singh Thank you. I will get back to you after trying recommended suggestions.
Thank you Subhash C. Juneja for the appreciation, privileged. This is because of back-stage performer Deepak N Modi whose high stringency leads to such quality approach and work :). Good Luck Poonam Mehta anytime :)