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

When I was trained on our Aria I thought that the wisdom was not to go above
an event rate of 5.
SRW


-----Original Message-----
From: Nebe-Von-Caron, G [mailto:g.nebe-von-caron@spdspark.com]
Sent: Fri 6/20/2008 7:21 AM
To: cyto-inbox
Subject: RE: Bad cell recovery post-sorting of human monocyte derived dendritic cells
What is the pH of your RPMI? As you do not control CO2 you would need to
stabilize is somehow, probably RPMI-hepes. Whilst serum adds essential
proteins and reduces stickiness it is also based on CO2 (bicarbonate)
buffering 

Sticking the stuff on ice will also effect your pH

In addition you have to test your osmolarity depending if it is designed
to be used with or without the addition of bicarbonate.




Gerhard Nebe-von-Caron 
Research Scientist and Biomedical Engineer 
SPDspark
Swiss Precision Diagnostics 
Priory Business Park 
Bedford, MK44 3UP, UK 
Mob+44(0)7792-116609
Tel+44(0)1234-835474 
Fax+44(0)1234-835006
mailto:g.nebe-von-caron@spdspark.com 


>From 
Tissue Engineering: From Cell Biology to Artificial Organs
by Will W. Minuth, Raimund Strehl, Karl Schumacher



3225	pH of the Medium
The maintenance of the acid/base equilibrium normally takes place using
sodium bicarbonate (sodium hydrogen carbonate), which serves both as a
buffering substance and as an essential nutritional component. An
increase of the CO: content results in a decrease in the pH value, which
is neutralized again by an increased content of sodium bicarbonate.
Finally, equilibrium is aimed at a physiological pH between 7.2 and 7.4.
The sodium bicarbonate buffer system in the culture medium consists of
NaHCO3, and CO2
NaHCO3, dissociated:	NaHCO3 + H2O <-> Na+ + HCO3- + H2O 
				Na+ + H2CO3-  + OH- <-> Na+ + H2O + CO2
+ OH-
This reaction depends on the partial pressure of CO2 in the atmosphere.
Under low CO2 partial pressure, the reaction equilibrium will move to
the right, which means the medium contains more OH- and is therefore
basic. Therefore, the sodium bicarbonate buffered media are gassed with
CO2 in the incubator. The buffering effect of the system is based on the
following reactions:

An increase in H+ causes:	 H+ + HCO3- <-> H2CO3 <-> CO2 + H2O 
An increase in OH-  causes:    OH- + H2CO3 <-> HCO3- +	H2O

Liquid media, which are used in a CO2 incubator, are usually held within
a defined pH range through the addition of sodium bicarbonate and the
controlled ventilation with CO2 via a control valve. If these media are
left for a longer time in culture dishes under a sterile hood, a pH
shift into the alkaline range can be noticed through the violet
discoloration. This originates from the fact that only about 0.3% CO2 is
present in the room atmosphere, whereas the incubator is usually
maintained at 5% CO2... If media must be used at room atmosphere, then
HEPES Buffer or another biological buffer is used for the stabilization
of the pH. In addition, there are culture media such as Leibowitz L15
that are equipped with a phosphate buffer system for working it the room
atmosphere

-----Original Message-----
From: Uriel TK [mailto:utk@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.