Bad cell recovery post-sorting of human monocyte derived dendritic cells

Ray Hicks rhicks at cytekdev.com
Sat Jun 21 05:38:14 EDT 2008


When my  cell counts were at odds with the sort count, it was most often due
to the users counting after spinning (as you say you're doing) or because
they were sticking to the tube (you don't say which plastic you're using) -
try using polypropylene tubes, to which most cells have a hard time adhering
(I've found these to be much more useful than serum-coated or silanised
glass or polystyrene)- and count the cells  before you spin them down -
spinning doesn't always go to completion even though you see a pellet there
may well be cells in suspension also you may well inadvertently resuspend
and discard a fraction of the pelleted cells (and in polystyrene tubes a
proportion of the cells may be forced into sticking to the bottom of the
tube).	It's simple to test if your loss is due to centrifugation - just
dilute some cells to the sorted concentration, pipette a known number into
some tubes, spin them and decant as you normally would after a sort, then
count them- if the loss is similar to what you've observed, voila! Count the
supernatant too, the missing cells are likely to be there, if not then use
microscope to see if they're sitting on the surface of the tube.

Ray

Ray Hicks
Cytek Development Inc
http://www.cytekdev.com
tel: +44 (0)208 1337 968 (UK/Europe)
fax: +44(0)208 5889 004
skype: ray.hicks.cytek 


-----Original Message-----
From: Uriel TK [mailto:utk at netvision.net.il] 
Sent: 18 June 2008 17:06
To: cyto-inbox
Subject: Bad cell recovery post-sorting of human monocyte derived dendritic
cells

Hello,
I'm having trouble with the cell recovery after sorting my DCs. I get 
less than 2/3 of the number of cells the machine counts as sorted. This 
is an ARIA (I). Yes, the same machine that made me go mad with 
frustration now behaves like a puppy, only that now the cells die after 
the sort - maybe there *IS* a connection here...

The facts are as follows: using 100 micron nozzle, at low pressure. The 
stream is stable all the time and accudrop is 100% pre and post sorting. 
running at 4-5k events per sec today, and ~2.5k previous two times, with 
a "rate" from 5 to 8. Efficiency was at >85% this time, and >90% 
previous two times. Sort logic is yield mask 8 and purity mask 16, 2 way 
sorting. The sorting takes 1 hour or less. I am trying to sort ~8x10^6 
cells presort, and have two populations that are ~20% of the total each. 
The sort yield reported by the machine (the number of events sorted), 
considering efficiency, is close to what would be expected, as is the 
total event count reported by the machine in comparison to my counts 
pre-sort. The post sort cells that I do get are the cells of interest. 
It's just that there are so few of them! I use sytox blue during sorting 
so i'm only sorting SB negative cells. The cells up until after the 
reading by the machine seem fine, with few % of SB positive, and OK 
FSC-SSC profiles, and the fluorescent intensities as expected, etc. I 
sort into 12x75 "FACS" tubes (plastic) with 0.5 mL FBS at the bottom the 
two previous times, and this time I diluted it beforehand with another 
0.5 mL of RPMI (I though maybe some kind of osmotic shock could be 
affecting the first cells to fall). The DCs presort are on RPMI, and the 
sheath fluid is sterile PBS (post sort I centrifuge and resuspend in 
RPMI to count). The all tubes in the ARIA are cooled (pre and post 
sort). Post-sort the DCs look OK, but it'd be very hard to detect any 
"pathology" by light microscopy with a 40x objective. I do get trypan 
blue positive cells, maybe as high as 15% or even 20%, which presort are 
<5%. I see a similar post-sort increase in the light scatter section 
where most of the dead and dying cells fall. In any case the cell counts 
I am reporting are only the TB negative. These are important, time 
consuming, and expensive experiments! It's really frustrating. Since the 
technical details seem to be right, I strongly suggest the issue is 
related to my DCs being fragile, as these are cells known for being 
delicate.

Do you have any suggestions?
Has someone else out there sorted immature and/or mature human monocyte 
derived DCs with success?

I looked at the archives but couldn't find anything specific to hmd-DCs. 
I've collected tips and ideas from other sorting threads, as well as 
some ideas of our own, and here they are in addition for your kind comments.

We're thinking of:
1) trying 15 PSI, but we don't know if the ARIA will be stable. Does 
anyone have experience with this?
2) "washing the tube" in FBS so that cells don't stick on it, as well as 
using 15 mL tubes to have a smaller surface to volume area.
3) I saw in a past post that "that you're not sorting onto the side of 
the tube, as the impact can kill the cells." Is this true? it will be 
very hard, or even impossible, to get the stream exactly in place!
4) Trying to reduce even more the time on ice, but that already is at 5 
hrs which is close to the "theoretical minimum" of a little less than 4 hrs.
5) maybe switching to glass tubes? any idea on the relevance of this?
6) reduce the serum to even less, and always have it pre-diluted in the 
target tubes.
7) Although all parameters seem OK, and other sort users are getting 
good results, we'll sort beads into slides/wells and count them before 
the next sort "just to make sure it is really working".
8) I'll resuspend the cells in PBS instead of RPMI to avoid the "medium 
shock" that has been described in the past. Or maybe use saline + HEPES 
if needed. The cells are in <=2 mL RPMI presort and end in >10 mL total 
medium. Any comments on this?
9) should I keep the flow rate at 2K anyway, or is the differential to 
5K not significant compared to the total PSI?
10) I'll try a "all sorted" gate with some cells to compare, and I"ll 
leave some of the pre-sort cells to compare as well.
11) I'll add FBS to the presorted cells as well.
12) the mAbs used to stain the cells have azide, but then most of them 
do and people sort that way without problems, no? it also gets diluted 
on staining and then at the post staining wash.

Many thanks for your help,

uriel.

-- 
Uriel Trahtemberg, M.D. M.Sc.
PhD student
The Laboratory for Cellular and Molecular Immunology
The Hebrew University - Hadassah Medical Organization
Jerusalem - ISRAEL

I am not a totally useless... at least I serve as a bad example.





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