I am trying to isolate neutrophils from rat whole blood, aprox. 5 mL collected via cardiac puncture into syringes containing 1mL ACD. I am pipetting whole blood directly onto 5 mL of Histopaque 1.077 and centrifuging for 40 min at 22C at 800g with 0 brake. I then pipet off the superntant layer of histopaque, plasma, and monocytes, and then proceed to RBC lysis of the lower RBC/granulocyte layer. My problems start there: the RBCs of rat blood seem to be almost impossible to lyse. I want to accomplish this without disturbing and activating the neutrophils, but no matter how many washes or lysis steps I include they seem to accumulate and will not fully lyse. Furthermore, I'm having a really hard time getting any sort of decent WBC pellet.
Sometimes I have to run several dozen samples at a time so I would rather not do a Histopaque 1119/1077 gradient because it is very time consuming to set up.
I have experimented with the histopaque 11191/077 gradient mentioned above, as well as dextran sedimentation with no success.
At this point I would be content with just developing a protocol that only lysis RBCs, leaving an enriched population of all leukocytes. I've had some success with using a lysis buffer and Restore buffer, followed by a wash with PBS and then ACK lysis buffer, but this seems like a lot of lysing steps that could potentially activate my nuetrophils.
I've been very successful with neutrophil isolation from cow and sheep whole blood using Percoll 1.084, but rat blood has given me a lot more headaches.
My downstream application is western blotting - my final step in the cell isolation protocol is lysis with RIPA Buffer + protease/phosphatase inhibitor.
Any help would be much appreciated!
Unni - is this the kind of media you're referring to? http://www.lonza.com/products-services/bio-research/cell-culture-products/reagents/cell-culture-reagents/lmphocyte-separation-medium-1077.aspx
And would I just replace the Histopaque with this media in my protocol?
I'm using lysis and restore buffers with the following formulations:
Lysis Buffer - Mix in 1 L DDI H2O: 2.83 g Na2HPO4․7H2O, 0.37 g NaH2PO4․H2O, Adjust the pH to 7.3 using Glacial Acetic Acid (carefully, one or two drops will be sufficient) or 1 M NaOH
Restore Buffer - Mix in 1 L DDI H2O, 2.83 g Na2HPO4․7H2O, 0.37 g NaH2PO4․H2O, 25.1g NaCl
Use 24 mL of Lysis buffer, mix and incubate for 90 seconds, then add 12 mL of Restore buffer.
Or this media?: https://www.cedarlanelabs.com/Reagents/Listing/Lympholyte_Special
Thank you so much for the protocol! Is the percoll gradient (steps 3-4) necessary? That sounds like a lot of work to prepare the gradient when I am trying to run 20+ samples alone...
have you thought about trying geyes lysis medium? it's a really gentle lysis buffer that doesn't do much damage to other cells. here's a paragraph from a paper showing that they used geyes lysis buffer to purify neutrophils from mouse. they give the formula, but if i remember correctly, it needs to be made fresh. actually you can make two solutions and mix them freshly, but the two solutions can stand for a long time. but i don't remember which chemicals went into which solution. perhaps someone else here knows that.
here's the paragraph:
Isolation of mouse neutrophils
Murine bone marrow was dispersed in HBSS (without Ca2+ and Mg2+) with 0.25% fatty acid-free bovine serum albumin (BSA) (HBSS/BSA) and was centrifuged (1256 g, 30 minutes, room temperature) over discontinuous 62–55% Percoll gradient in HBSS. Mature neutrophils were obtained from the interface (purity, 75–85% by cytospin), and contaminating red blood cells were removed by Geyes lysis (130.8 mM NH4Cl, 5.0 mM KCl, 0.78 mM Na2HPO4, 0.17 mM KH2PO4, 5.56 mM glucose, 1.04 mM MgCl2.6H2O, 0.28 mM MgSO4.7H2O, 1.53 mM CaCl2.2H2O and 13.39 mM NaHCO3); cells were washed three times in HBSS/BSA. Neutrophils were resuspended in D-PBS with Ca2+ and Mg2+ (D-PBS+) supplemented with 1 g/l glucose, 4 mM sodium bicarbonate (D-PBS++) for assays.
and here's the reference to the paper. perhaps writing to the authors would work.
http://europepmc.org/articles/PMC3104032
cheers polly
try this:
Laboratory
Animals
(1979) 13,329-331
Haemolysis
of
various
mammalian
erythrocytes
in
sodium
chloride,
glucose
and
phosphate-buffer
solutions
TOSHIAKI
MATSUZAWA·
just tell me if it works!!
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