use BD lysing buffer to lysis the RBCs from any of the sample including blood when u r isolating PBMcs from the blood. Make 1X FROM 10X by water and use 3 times as the volume of your suspension.

make sample containg 1 x 10 7cells then centrifuge at 400xg, 4degree,

resuspend cells with 5 ml dulbecco phosph bufferd saline (without ca and mg) centrifuge same speed, remove cells and resuspend in 2 ml of BD Pharm lyse lysing buffer , incubate at 2 min at room temp, this helps in lysing RBCs

Lysing buffers should work, but could impact cell phenotype. Since AM are very adherent to plastic, you would probably be ok if you just plated your cells, let them adhere , then washed a couple times with PBS or culture media (the next day).

We made some good experience with Qiagen, Buffer EL Erythrocyte Lysis Buffer, CatNr 79217. 'Was also feasible when preparing bone marrow stem cells and seemed not to interfere with any parameter measured. Likewise, it worked when we prepared vascular cells. Good luck.

I agree with Joshua, when culturing macrophages you can always let the cells sit for a couple of hours on a petri dish, and then wash away the suspension cells. This method would also make the macrophage population purer since you would get rid of potentially "contaminating" cells that reside in the suspension of the BAL.

However, if you insist on RBC lysis, I would recommend to use Ammonium Chloride solution. I use this method when isolating granulocytes etc. from bone marrow, and it does not activate the cells immunologially, but as Joshua said, you never know if it might affect the cells in other ways. This lysis buffer is also cheaper than using the company kits, but it still works like a charm.

Ammonium Chloride Recipe:

0.15 M NH4Cl (8.26g/l)

10 mM KHCO3 (1g/l)

0.1 mM EDTA

pH 7.4

(I would recommend that you filter the solution before using it)

Using this method you should first resuspend the cells in PBS (at least once), spin the cells to a pellet in a 4 degree C centrifuge. Resuspend in 5-10 mL of the Ammonium Chloride solution and spin them down again for 5 min. Discard the ammonium chloride solution and wash the cells at least twice in (preferably FCS/FBS containing-) media.

Good luck!

I've regularly flushed the bone marrow from the long bones of mice and find that I also had some RBCs that I wanted to get rid of. A quick, easy and cheap way I use to lyse these RBCs is to treat this cell suspension with buffered water.

I make the buffered water with 9.7mL ddH2O and 0.3mL 1M HEPES. I then add 9mL of this buffered water solution to the cells for 45-60 seconds and then make isotonic by adding 1mL of 10xPBS. You can then spin these cells down and re-suspend them in your buffer. This appears to have minimal effect on the cells viability, morphology and function and I have successfully cultured cells from these preparations.

The only worry with ammonium chloride solution (any any chemical lysis solution) is that it may activate certain cells types as Sebastian says. Trial and error time!

Use sterile distill water and give a quick wash with it to your cells (apprx. 20 seconds) and dilute it with PBS then. Centrifuge ur cell suspension and plate then.

When we culture primary macrophages, we plate out the isolated PBMC and mature the monocytes for a total of 6 days, washing away excess cells 2 or 3 times during that time when the cells are fed with fresh media. As several answers above have said, these cells are very adherent to plastic and this results in a relatively pure macrophage population. Be aware that plastic also activates the cells, so if you are used to cells which have matured on glass, the plastic might give you different results. However, the greatest variation in your results will come from using primary cells, as cells from different donors can vary widely in their reactions.

We get rid of the RBC during the PBMC processing by using 1X RBC lysis buffer, our recipe is below

RBC Lysing Solution (10x) – In 1L dH2O add

NH4Cl, 80.2 g

NaHCO3, 8.4 g,

EDTA disodium, 3.7 g

filter solution and store at 4°C.

Dilute 1:10 in dH2O to make working solution

We also often use an ammonium chloride RBC lysis during our PBMC isolation, and due to running a lot of PBMC isolations I have tested its effects on macrophage activation for HIV infection, cytokine production and phagocytosis and anecdotally it does not change my results. When we do RBC lysis, we still get a number of other cell types, so for a pure population I agree with the above commenters, adherence and washing will be your best bet.

we generally allow macrophages to adhere then change media to remove non-adherant cells. Washing multiple times will reduce RBC contamination.

FMost of the methods described here (except the one by Piers Gordon-Brown) are far too complicated and expensive all these kits etc. "Hypotonic lysis" is the old technique used to isolate leukocytes w/o contaminations from RBCs. It also work with slight variations for other cells, e.g alveolar macrophages. After the "last" centrifugation, re-suspend the mixed cell populations (e.g., WBCs, macrophages or what have you + RBCs) gently in 9 volumes cell culture grade (endotoxin free, sterile) water. I do not like the buffered water solution, it still has too much (30 mM) HEPES. After 45 second or so (herein lies the variability depending on the cell type, you will have to determine the exact time depending on your cells), add 1 volume of 10 x PBS. Mix gently and the spin gently (400- 600 rpm for 5 min). Resuspend the pellet in your favorite complete culture count and determine viability.