Problem with RBC lysis - The darn things won't lyse! (Aug/08/2005 )

I've been trying to get the following reagent to lyse RBCs. I know it's worked before in my old lab, but since moving to this new lab it just doesn't work:


Lysing solution (courtesy of the Columbia University Flow Cytometry Lab)

Ammonium chloride (NH4Cl) 0.15M 8.29 g
Potassium bicarbonate (KHCO3) 10mM 1 g
EDTA 0.1mM 0.037 g
H2O dist 1 l
filtered with 0.45µm filter, stored at RT.



I doubt my Ammonium chloride or Potassium bicarbonate are 'bad'. I'm wondering if it's the EDTA. I'm using 8.0 pH 0.5M EDTA solution from Fisher. I'm just diluting it down to the proper final concentration. Does EDTA solution go 'bad'?

I'm thinking of buying the powdered disodium salt of EDTA and making up a fresh batch and then seeing if I'm still having a problem with RBCs not lysing.

Does anyone have any other pointers?

Thanks!

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I use this recipe for 1L

8.99g NH4Cl
1g KHCO3
40ul 0.5M EDTA
adjusted to pH7.3.

Works every time

The EDTA you are using should be fine

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My recipe of RBC lysis buffer:

0.32M Sucrose
5mM MgCl2
10% Triton X-100
10mM Tris-HCl, pH 7.8

It works fine for me.

Hope it helps.

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"my" recipe is very similar to yours, works always for me...

150 mM NH4Cl
1 mM KHCO3
0.1 mM EDTA
pH 7.2 –7.4
filtered with 0.45µm filter, stored at 4°C, brought to RT before use.

Cells are incubated for 5 minutes in buffer, then centrifuged at 300g for 5 minutes...

mike

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Thanks guys!

I think the culprit is pH. I measured my RBC Lysis Buffer and it was at a pH of 8.4.

I measured the Fisher 0.5M EDTA solution and it was at a pH of 8.22 (it is labled pH 8.0 +/- 0.01). So, not only is my prepackaged EDTA solution off, but the the RBC Lysis Buffer is even more off once mixed.

I measured the Gentra RBC Lysis buffer, and it reads at a pH of 6.47.

The Gentra one works great, so I'm wondering... many folks here have stated that they adjust their pH to around 7.2-7.4. If Gentra's is 6.47, wouldn't that be a better adjustment to make?

I suppose I could just test both pH levels and see what works best.


Time to go order some concentrated HCl.

Thanks a bunch!

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Mine is used for RNA so has to be isotonic

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Well, I tried two different pH adjustments. One at 7.2, and another at 6.47. Neither solutions caused RBC lysis.

I tested the Fisher EDTA solution, and it's measuring at a pH of 8.3. It's supposed to be 8.0. I'm wondering if I received a bad batch.

I'm ordering crystalline EDTA now, and will try another batch of buffer with that.

If that doesn't work, then it means either the Ammonium chloride is bad, or the Potassium bicarbonate is bad.... or, it means that for some reason the above RBC lysis buffer recipe doesn't work in Louisiana.

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