I tried isolation of chicken monocytes from blood using Histopaque 1.077 or 1.083 and centrifugation 800 xg for 15 min, washing by PBS 3 times at 200 xg. then culture in plastic dishes for 1 h at 39 C and washing non-adherent cells.
the problem is that there are many adherent thrombocytes, how to get rid off these contaminating thrombocytes?
What anticoagulant are you using? Citrate is usually used to keep samples 'calm' for coag and platelet function testing. Also the usual caveats for blood samples, especially if you're finding problems: anticoagulate immediately, and process immediately before the cells start to react to the ex-vivo environment.
Sorry I forgot to mention this. I collect blood by a syringe contains 3ml PBS with 3mg per ml EDTA, and aspirate 3 ml blood.
Problem of chicken blood is the nucleated platelets with nearly same density of pmncs, thus iam considering to try slow spinning after agglutimation of platelets by methyl cellulose or dextran.
I will consider citrate as anticoagulant in conditions adjustment.
Thank you very much
Hello Moustafa.
Right now Im starting to work with chicken PBMCs and just as you mentioned the biggest problem I'm facing is the thrombocytes and red blood cell contamination. I have used Ficoll-paque 1.077 and tried several lysis procedures with a commercially available RBC lysis buffer and 0.2% NaCl solution, but with both I'm still struggling with the elimination of red blood cells. But what concerns me the most is the thrombocytes contamination, did you find out any solution to this problem? @Moustafa Mohammed El-hamouly
Hello Gabriela Avila Morales,
The best way is to collect peritoneal macrophages, 2-4 days after intraabdominal injection of starch or sephadex. Then purification by centrifugation on histopaque 1.083 to remove heterophils, then adhesion to plastic culture dishes to remove lymphocytes. purity was more than 95%. or to use HD11 macrophages if available.
I tried several times to isolate PBMCs by several methods, except FACS or dynabeads may be applicable despite they cost and interfere with cells by the used antibodies.
Notes may be helpful:
1- In my trials to isolate PBMCs after histopaque (1.077 or 1.083) with centrifugation (600-1200 x g) always cells were contaminated with many thrombocytes, which did not die even after 5 days incubation at low and high FBS%. Thrombocytes attached as fast as monocytes., and did not detach by repeated washing.
2: Trials to aggregate thrombocytes with dextran or methyl-cellulose then slow centrifugation (25-100 x g) also failed.
3. Using EDTA or Heparin had no effect.
Finally, I hope you find the best way to isolate monocytes, I hope to hear good news in your experiment, best wishes.
- Badges
- Science topic
- Similar topics
- Microbiology
- Assays