Hi, I am stuck in trophoblast cell isolation from the human term placenta. i have followed different protocol collected from articles. the main problems i am facing are during trypsin digestion, the digested supernatant becoming very slimy and difficult to filter using 100um and 40 um nylon mesh consecutively. though extra dnase powder is somewhat helpful. the other problem is during percoll gradient. i am using 90%isopercoll in pbs, tried 70%-5% discontinuous and 40% and 25% percoll. i got success with only two gradient using HTR8 cells and got a cell layer in the middle of the two percoll gradient. however, later, i could not make it again. and microbeads shows that (1.02, 1.04 and 1.13). i am using 1200Xg, 30 min centrifugation without brake. please suggest me to overcome the protocol. i will appreciate if anyone can provide me detail potocol as well. i am preparing the gradient using pipette, slowly discharging the liquid on the top of the higher density, with a round motion of the tip, touching the centrifuge tube wall.
thanks in advance.
Dear Jaganmoy.
I would like to help you more precisely, but I work with trophoblast cell lines and Toxoplasma gondii infection (BeWo/HTR-8) and unfortunatelly I don't have experience with primary trophoblast isolation.
Although this, along the years I follow and have read the papers from the Martin Knöfler's group. He and his partner Sandra Haider have extensive experience with trophoblast isolation, and in reason this, I send to you the paper that follows in this my comment (Haider et al. 2016 PNAS), which described the isolation of some populations of trophoblast cells from human first trimester placenta, and this paper cited some base references that also could help you. But, I understood that you want to isolate trophoblasts from human term placenta, not from first trimerster, and therefore I'm send to you another paper, very recent (January 2021), that I'm studying, in which the authors made the isolation of human primary trophoblasts (PHT) from human term placenta. Maybe this paper can to assist you on some way (Shao et al. 2021). Despite this, the Professor Knöfler has a recognized experience in this field of trophoblast isolation, and his group is a reference on trophoblast biology in the last decades. To reinforce, I send to you one more paper (Haider et al. 2014) in which the isolation of term placenta trophoblast was made (as you need to make).
Isolation of trophoblast seems to be very difficult, and I'm sending positive energy for your success.
I hope that I helped you.
Best wishes!
thanks Marcos, I have already gone through the articles you have sent. However, I dont understand, why the cell layer is not coming down after the centrifugation process.
thanks again for your kind reply.
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Virology
- Virus