Dee, summary macrophage, monocye harvesting

Dee Harrison-Findik duygu.harrison-findik at imvs.sa.gov.au
Mon Mar 11 22:35:13 EST 2002


Dear Flowers
About two weeks ago, I have posted questions regarding harvesting adhered
monocyte/macrophages in a viable and efficient way.
I have received many responses and have already sent a general thank you
message to the responders through this site .
In between, I have forwarded all the replies to the Flowers who personally
requested them.
I still prefer to forward the original replies to people who are interested
because summaries are afterall personal interpretations.

However a brief summary of the suggestions are:

 - Using reagents beside trypsin like accutase (Phoenix flow systems) or
lignocain (any hospital pharmacy or Phoenix Pharmaceuticals).
Concentrations not reported.

- Jagannath Chinnaswamy [Chinnaswamy.Jagannath at uth.tmc.edu suggested a non
enzymatic buffer from GIBCO-BRL but has not sent any more details.

-If isolating them by adherence then the adhering time should be kept to
minimum (20-30 mins. without serum)

- They can also be plated on large petri dishes that are not tissue culture
treated.  Wash the adhered monocytes by cold PBS (without calcium and maybe
add some EDTA).  This response was mailed to the cytometry site by Ronald
Rabin.

- Majority of the responders recommend to keep the cells on ice (about 15
min. with some EDTA)  before harvesting them.  Harvesting by PBS/ EDTA (no
calcium) is recommended (compared to trypsin) if the cells are going to be
stained by antibodies straightaway and EDTA should be washed off carefully
before the antibody is added.
- The responses regarding scraping them off the plates are conflicting.

-Olinda Assis [oamfilho at cpqrr.fiocruz.br] has told that one of his students
has cultured monocytes in polypropylene tubes to avoid attachment.

I hope this helps.
Dee







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