Hello, is there any experience to isolate mouse lung endothelial cells using MACS?
I have troubles about viability and purity about the cells.
I have tried to isolate and culture mouse lung endothelial cells from C57BL/6J mouse 3-4weeks.
I isolated the cells using MACS with CD31 antibody double sorting.
but the viability was too low after isolation and get too low amount of cells.
even I used 5 mouse but after isolation, only about 10% conflunecy was left in 25T flask.
I tried to change dissosiation enzyme from collagenase 2 to 1.
And also I tried to use concentration of collagense 1 from 1mg/ml to 3mg/ml in 25ml volume for 4-5 mouse. but all was failed.
there are another problem about endothelial cell purity.
after isolating the cells, the purity of endothelial cells were confirmed by FACS using CD31-APC antibody and there were not much endothelial cell. The purity was sometimes 60% and sometimes 4%. So now I try to isolate doubly CD31 and CD102 using MACS and wait the results.
give me the help, sincerely.
Tips for isolating mouse lung endothelial cells (MLECs) using MACS: 1. Optimize lung processing and enzymatic digestion. 2. Use specific endothelial markers (CD31, CD144, VE-cadherin). 3. Adjust antibody concentration and incubation time. 4. Choose the right MACS column and separation strategy. 5. Use endothelial-specific media and maintain optimal culture conditions. Consider: - Negative selection to remove non-endothelial cells - Combining markers for improved specificity - Gentle cell handling to minimize shear stress
Junyeol Han
Wong E, Nguyen N, Hellman J. Isolation of Primary Mouse Lung Endothelial Cells. J Vis Exp. 2021 Nov 10;(177):10.3791/63253. doi: 10.3791/63253. PMID: 34842233; PMCID: PMC11101010.
Very Thanks for your answer.
I've considered the tips you mentioned.
I tried enzyme and its concentration variably. I've never passed enzyme incubation time over 45mins .
I also tried negative selection like CD45 and CD326.
I have used DYNABEADS FLOWCOMP FLEXI (thermo fisher) and label antibodies.
I tried to change gelatin concentration coated to culture plate.
I've used culture medium, Dulbecco’s modified eagle medium (DMEM), high glucose, 20% heat-inactivated FBS, 30 μg/ml ECGS, 20 U/ml heparin, nonessential amino acids (NEAA), and penicillin/streptomycin (P/S).
actually I thought I consider lots of things several articles mentioned.
but I don't know what is the problem :(
Thank you for your response. However, the issue has been somewhat resolved, so I appreciate your suggestions but will have to decline them. Wishing you all the best in your research.