Hi Michael!

This sounds strange to me. Probably, there is something wrong with your trypan blue. Did you culture the cells anyway?

In my I hands I use 40µm strainer and no enzymatic digestion. Afterwards, pipetting up and down is also essentiell.

All the best,

Claudia

Maybe your cells aren't as dead as you think they are? Have you tried looking at your recovered cells in flow cytometry (using PI or DAPI)? I find flow is a way more reliable measure of viability than trypan counters.

Other than that, I might suggest tamping down the ACK buffer a bit. It's pretty harsh on all cell types in the spleen so you might increase the survival of your monocytes if you use less ACK buffer. I've found in the past that, when doing mouse splenocyte isolation, I've had to ease back on the ACK buffer a bit compared to what the manufacturer protocol says.

It could also be the mouse itself. It sounds like you've tried this a few times - are they always around the same age? I get much lower yields of splenocytes when the mice are really young. If you haven't been already, try to make sure your mice are at least 8 weeks old.

To me, ACK buffer is most suspicious. Because of it's lysis character, the buffer need to be manipulated carefully, or it will kill all the cells. Find out the manual of this buffer from suppliers' website, or ring their technique support maybe helpful as well. Most RBL buffer is formulated for whole blood, the components, such as serum, is different from spleen cell mixture, this may change the potency of the buffer.

Claudia, Govinda, Shiqiu, many thanks. I did try to culture the monocytes, they're definitely dead.

Less ACK lysis sounds like my best bet.

The mice are usually retired breeders, so around 6-9 months old. Perhaps that's too old.

Hi all,

I solved this issue. Here's a protocol that works, avoid ACK lysis.

Checklist

Cool PBS without Ca or Mg

Cool 50 ml tube

Cool PBS without Ca or Mg with 1 mM EDTA

Cool PBS without Ca or Mg with 1 mM EDTA, 2% FCS

Cool 15 ml conicals

Cool 5 ml magnet tubes, and cool the magnet

Actions

Resect mouse spleens

Force mouse spleens through a 100 micron filter with a syringe plunger (cold PBS without Ca or Mg with 1 mM EDTA) on ice

Wash spleens with cold PBS without Ca or Mg with 1 mM EDTA, 2% FCS

Use stemcell mouse monocyte isolation kit (19861)

Yield: about 1.5 million monocytes from spleens