Summary Document

This document is a summary of message from the Purdue University Cytometry Laboratories Cytometry Discussion list.

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Dear all,

A very big thank you to everybody that replied to my query about staining in
Eppendorf tubes.

Below I have collated all the answer received.

I think the suggestion to have a couple (or more if needed) test on
speed/time and pellet appearance is correct, and I'll pass the message to
the PI interested. Plus I'll do a couple of test myself.

Thank you very much again for your help (as usual).

Mara

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Responses:

Staining in microfuge/eppendorf tubes can really speed up your processing.

We routinely stain 100,000 to 1,000,000 cells in <100ul ; and spins are
about 14-18sec at 10,000g (depending on cell type, fixed or permed, etc).

Compared to 5-8minutes in the 5ml "FACS tubes"- it's a nice change.

(I will say- once you have more than 10-15 tubes to process simultaneously;
time to look at staining in a 96 well plate- unless you have stainless steel
thumbs!)

IMHO- it's best to work out the speed/timing for your specific
rotor/microfuge combo, and your particular cells. (You didn't mention what
type cells..)

For instance- when processing HUVEC, my spins are a shorter than when I
stain PBMC's as the HUVEC's pellet faster.

Aliquot your cells into a couple of tubes, then compare recovery/"integrity"
(are the cells easily resuspended- or now crammed into a tight snot ball?)

Ok, not the most scientific description- but a visual you will get right
away if it happens... I've used 3 different microfuge's and found they
each had a "sweet spot" for best wash/ recovery. {An extra few seconds on
one made a nice "PBMC puree"- even though I thought I had the same g
force...}

I like the 2ml snap caps (I think I got from ISC?) instead of the
"traditional" 1.7ml because: 1) the extra volume for washing and 2) the
cell pellet resuspends quite nicely.

If you prefer the longer/slower spins of a benchtop (600g, 5min) you could
still do that in a microfuge tube. We've even set up mini-l-ficolls with
200ul blood!

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Since the ImageStream uses Eppendorf tubes as the standard sample container,
we have quite a bit of experience with the format. I forwarded your query to
our bio group and this is what Tad had to say:

If using a minifuge, we spin cells at 300xg for 5-10 minutes (same as in the
benchtop larger centrifuges) for all sizes of tubes. Check the minifuges
instruction manual or website to determine what that translates to in terms
of rpm, as this number depends on the rotor radius. Avoid spinning really
hard for short times as this potentially damages the cells.

In terms of staining in eppendorfs, it is important that the wash steps
dilute the staining reaction 10 fold, so if you are staining in 100 uL
volume, you will want the add about 1 ml of wash buffer prior to the spin
(in this case use 1.5 ml tubes). We typically stain cells at concentrations
between 1 and 3 x10^7 cells per ml (ie 1-3x10^6 cells per 100 ul).

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We do all sorts of staining in eppendorf tubes (tetramer, IC, etc). We use
1.5 ml tubes, and colorless tubes are best to see the pellet. We spin in
benchtop microfuges for 3-5 min at 2000 rpm, and sometimes I'll cheat and
use the "quick- spin" microfuges for 10-15 seconds if I have only a few
tubes. Good luck!

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I had to do this a while ago because our standard centrifuge had broken
down. I used 1.5 ml tubes. I believe speed was around 200 rpm, but you could
easily calculate that from the actual radius.

You cannot decant as easily, I used cautious suctioning.

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I use 2 ml Eppendorf tubes for staining MDCK (dog kidney) cells regularly
using a swing out bucket.

To treat the cells as gently as posible I use 60 to 100 g for 10 to 15
minutes (4 °C). But with many kits 300 g for 10 minutes is suggested which
works, too. But sometimes 5 minutes at 300 g is not enough to spin all the
cells down.

Starting at fixation of 1 million cells in ethanol and 6 centrifugation
steps i still end up with enough cells in 1 ml or 0.5 ml to measure 10000
single cells easily. Ah, the ethanol fixation is done in 15 ml Falcons but I
switch to Eppis after two centrifugations for 30 min at 100 g.

Well, this is how I do it and it works, at least with my samples. :-) Let me
know wether it works with your samples as well.

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I use 1.5ml epi tubes. I stain in 100 to 200ul volumes. and wash with 1ml.
It works fine.

For normal surface markers I spin at 300rcf. for 3.5 to 5minutes. Depends
on how much of a hurry I am in. For intracellular staining I usually spin
faster at 700-1000 rcf. Cells get very buoyant when they are fixed,
especially in BD's cytofix/cytoperm fixation/wash solutions.

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I just finished working up a procedure for doing indirect immunostaining on
oocytes from an indigenous mussel from New Zealand, and I used either 0.6 or
1.5 ul eppendorf tubes.

I spun at 0.1 or 0.2 rcf units for 15 or 30 seconds (30 semed to get the
ocytes to tbe bottom of the tube better) using the little benchtop eppendorf
microcentrifuge that looks like a half-dome.

If I gently poured the liquid out of the tubes, the cells would stay behind.

I used a low speed because these cells are quite mechanically sensitive.
Ther are also quite big cells, so they may pellet more easily. They are
about 50 micrometers in diameter.

It worked well.

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We routinely stain tissue culture cells and whole blood samples using a
refrigerated microfuge. We use 1.5 ml snap-cap tubes, With the speed
limited to 10,000 RPM (max), we spin for a total of 12 sec- it takes our
microfuge about six seconds to get up to max speed. The microfuge allows you
to remove most of the supernatant liquid, so antibody concentrations are
more reproducible.

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I have used 1.5 ml tubes and spun at 2,000 rpm for 4 to 5 min. It works fine.

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For what it is worth, I always stain in eppendorfs. 1.5 or 2.0 ml, and I
spin for 1 minute at 4000 rpm.

Sometimes I spin a little longer if I'm staining few cells and don't expect
to see a pellet.

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I've always used 1.5 mL epi tubes. I stain my cells for about 30 minutes on
ice (in closed bucket). Then I wash the cells with 250 ul of cold buffer
and centrifuge at 13,000 g for 5 minutes. After the spin I generally remove
all supernatant with a thin disposable transfer pipet, take the pellet up in
300-350 ul of cold buffer and dispense the sample into a 3 mL FACScan tube.
Although it's worked well for us, I hope this helps!

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I have done this with a standard 100 ul containing 1 million cells (10^6)
format, washing with 1 ml of buffer. I use 1.5 ml Eppi tubes.

A nice thing about it is that you can pellet the cells at 7500 RPM for 15
seconds.

Try it on some trial samples first, to make sure your cell survive that
G-force. Most do fine in my hands though.

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I always use a minifuge. I use the 1.5 to 2 ml tubes to allow for addition
of wash buffer in washing steps (1 ml of wash buffer for each step) and
centrifuge at 2500 rpm or 350 g each time. It gives good results and you
can see the pellet through the tube walls after each wash step if you choose
translucent tubes.

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