I think you have two problems: your cell dissociation step is not effective and the cells coming out of that step are prone to clumping, possibly because they are half-dead from scraping. In my experience, macrophage lawns are best detached by rinsing the lawn twice with ICE-COLD PBS + 2.5mM EDTA and then incubating the plate ON ICE with ice-cold PBS + 2.5mM EDTA for 10-15 minutes. [EDTA is notoriously hard to get into solution, so you need to make a 0.5M stock solution at pH 8.0 always stirring while adding EDTA and keeping the pH at 8.0].

Look at the cells after 10-15 minutes in ice-cold PBS + 2.5mM EDTA and if they have rounded up, they are ready for scraping with a rubber policeman. Scrape the plate over a black surface - you should see the lawn detaching beautifully. The next steps are very important: these cells are quite stressed, with the EDTA, lack of serum and ice-cold temperature shock. You need to quench the cells that have come off the plate in a large volume of refrigerator-cold (i.e. 4'C) HBSS without divalent cations containing 2% Fetal Calf Serum. Keep the FCS present in all subsequent steps. Use HBSS without Phenol red for flow cytometry applications. The cells should have excellent viability and not be clumped. You should not need to undertake any filtration step.

Dear Steven,

Clumping and/or the formation of aggregates can be a severe problem, as you have noticed. But it depends on what you want. If you can live with the fact that a number of observations are due to clumping, I would suggest to go to the FS/SS page of your cytometer and gate the single cell events. Eventually, you can use pulse time as a second gating parameter to eliminate clumps.

This is what I would do, rather then to engage in cell dispersal methods which may introduce new and unknown artifacts.

Greetings,

Harrie

Hi Harrie,

Thank you for your response. I agree that gating is a valuable tool and could be a valuable way to isolate a cell population (single cells in this case). However, I am concerned that the clumping of my culture macrophages will block or jam the FACS if I can't get them through a 40 micron filter shortly after they are dissociated.

Mild EDTA will be good option to chose. If you are not willing to use mild EDTA, you can try some DNase. Also, cultured cells does not often block FACS machine (if it does at some point try running some bleach in your instrument - then water - sample). Depending on what platform you using for acquisition (e.g. Diva) you can use FSC-A/FSC-H based gating for getting rid of clumps of cells. I hope it helps. Good luck!

If I understand it correctly you resuspend the cells after detaching in DPBS. For some primary cells we had similar problems (after cell detachment and a following centrifugation step the cells started to clump). We solved the problem by adding a small percentage of FCS (1-2%) to the buffer.

Sharad,

Thank you for your suggestions. A solution of DPBS with mild EDTA (5mM) is going to be involved during my next attempt.

Tobias,

I was under the impression that adding serum is more likely to introduce Ca/Mg to the Ca/Mg-free PBS solution (supporting cation induced cell-cell adhesion) and contains adhesion factors. But if protein support has aided you, than I will try a solution containing 1-2% BSA (more defined).

I appreciate the feedback!

Enzymes/detaching reagents may promote cell clumping for various reasons. Simply use gentle scraping with plastic scraper. Sure, your cells will be a bit damaged, but not too much. We normally see 90-95% viability after this procedure and never see clumps. Using EDTA and protein-containing FACS buffer also helps to prevent further scraping.

SO far you got only good advices, but let me summarize and add some more ;)

1. Discard medium from culture and add PBS w/o Ca and MG, but with EDTA added. Left 5 minutes on RT, discard and add your non-enzymatic solution, However, I suggest you to try Accutase, which is very gentle enzymatic solution. You can left solution on cells even for 1h with no harm, usually 30 min is sufficient. Collect floating cells, and wash 2x with PBS + EDTA. I don't like scrapping, gentle enzymatic solution is my preffered way. And it is quicker if you have large number of wells to collect, not mention collection from 24-well plates, or even 48-well plates.

2. Up to the top in tube add PBS + 2 % inactivated FBS + 2mM EDTA (let's call it Wash/Staining Buffer - WSB) and wash cells. If you expect certain number of dead cells, you my add some DNAse.

Moreover, you can use Accumax, solution distributed by eBioscience, same as Accutase, to prevent cell clumping.

3. Once you peleted cells, discard supernatant, and first gently tap few times bottom of your tube(s) to loose pelet. Then use pipette, tak 1 ml of WSB and resuspend gently pellet. Add another 2 ml and repeat. If your cells are picked from wells that represent single sample for flow cytometry, just lower the ammount of WSB.

4. All the time while cells are alive, i.e. not fixed, don't ever use vortex !!!!

5. For all the staining procedure, use polypropilene tubes, not polystirene. If you need to use polystirene tubes for acquisition on cytometer, transfer cells from polypropilene tubes just before acquisition.

6. Centrifugation force for large cells like macrophages should be 200-300g,

7. Use FSC-W vs FSC-A or vs SSC-A to exclude clumps in tube from analysis later.

Good luck !!

Denis

Steven,

Along with adding some serum, how about trying pipetting for few times. I think you wont lose cells. Gentle pipetting would segregate cells effectively. Hope this helps.

best of luck,

Prakash

Agree with suggestions above. I use 2 mM EDTA in the buffer and it helps to prevent aggregates during flow. Depending on your FACS machine, fIltering is usually not necessary for cultured cells. Exclusion using FS/SS during analysis would also help.

I have read the answers and all of them are very good; however, I use a solution to detach the cells and avoid clumping which is PBS free of Ca/Mg 1 % gelatin and 2 mM EDTA. To endure detachment and centrifuging use cold PBS gelatin and flor stainging and fro suspension of cells prior flow cytometry use the solution without gelatin

good luck

To add to the above, grow BMs in petri dishes instead of tissue culture dishes. Wash in cold PBS and then lift in cold PBS, 5 mM EDTA 5-10 min and then gently by pipetting around the dish. For a 15 cm use 10 mLs of PBS/EDTA. We use the Sigma none-enzymatic cell dissociation as an alternative with good access.

I think you have two problems: your cell dissociation step is not effective and the cells coming out of that step are prone to clumping, possibly because they are half-dead from scraping. In my experience, macrophage lawns are best detached by rinsing the lawn twice with ICE-COLD PBS + 2.5mM EDTA and then incubating the plate ON ICE with ice-cold PBS + 2.5mM EDTA for 10-15 minutes. [EDTA is notoriously hard to get into solution, so you need to make a 0.5M stock solution at pH 8.0 always stirring while adding EDTA and keeping the pH at 8.0].

Look at the cells after 10-15 minutes in ice-cold PBS + 2.5mM EDTA and if they have rounded up, they are ready for scraping with a rubber policeman. Scrape the plate over a black surface - you should see the lawn detaching beautifully. The next steps are very important: these cells are quite stressed, with the EDTA, lack of serum and ice-cold temperature shock. You need to quench the cells that have come off the plate in a large volume of refrigerator-cold (i.e. 4'C) HBSS without divalent cations containing 2% Fetal Calf Serum. Keep the FCS present in all subsequent steps. Use HBSS without Phenol red for flow cytometry applications. The cells should have excellent viability and not be clumped. You should not need to undertake any filtration step.

Hi Steven,

I'm not used to work with BM derived Macrophages, but I usually differentiated human macrophages from monocytes. At day 7, I detach the cells using accutase solution (Millipore). Briefly, I previously wash with PBS in order to remove some sera components which can inactivate accutase (and also saving the initially/after PBS wash dettached macrophages), and after I leave remaining macrophages 30' at 37ºC with accutase solution (3 mL - 10 cm diameter plate). After this step, I can recover > 90% of attached macrophages, without affecting viability. When I acquire in FACS, I don't have cell clusters, but I agree that you can use EDTA as antiaggregating agent in order to avoid cell clumping to improve this point.

Surface antigens and viablility are not modified, and recovery yield is really good.

As I have explained, I don't know whether my model could be comparable with BM derived macrophages differentiation, but I hope it will helps you anyway.

Good luck!

Hi Steven

I have used both EDTA buffer and FBS to prevent clumping for different adherent cell types and both worked. You may try FBS 2-3%

Regards

Hi Steven

Have you used any Leighton tubes and adhere on the microcoverslips placed in the leighton ,grow the cell on the leighton tubes for certain duration of time such as 10 15 min and wash the cells gently with cold PBS to dislodge unwanted cells and then use the harvesting solutions that you use for the recovery.then check the viability of the cells and count the cells .That way you know the percent recovery and get the cell concentration of your choice.

Thanks

Thank you everyone for the insightful responses! I have a few new methods to try.

Try to use the tubes I refered

One thing we ALWAYS do with samples presenting this kind of difficulties is adding a bit of DNaseI . Often it turns out clumps are generated by long DNA strands liberated from dead cells, and trapping loads of, otherwise, live cells. Breaking up this "net" of DNA strands often helps. Also, do this before filtering, of course, and limit centrifugation to absolute minimum time and speeds ...

HTH,

Jan

Hi Sharad, Denis and Jan;

What concentration of DNase do you add?

I don't remember off the top of my head. Will have to look that up next Monday ... What I do remember is that we prepare stocks according to Maniatis, and add minimal amounts of that, most likely a few microliters per sample (typically around 300 ul) ... More info Monday !

Jan

We routinely use BMM and culture them in non-tissue culture treated petri dishes. Cells can be released for FACS or replating by simply adding 4C sterile PBS, incubate at 4C for 10min and then scraping cells with soft silicone/rubber cell scrapers. As you said, under hemocytometer we don't see a large degree of cell clumping. My former lab has protocols for FACS analysis of BMM if the suggestions above don't solve your problem.

I totally agree with Jan Bayer, DNAse I might very well be the solution to your problem, in most cases clumping is the result of broken nuclei and release of high molecular weight genomic DNA: in "my" protocol you resuspend the cells in the following buffer:

20ml FCS,

1000U Heparin (Sigma#H3149),

25mg DNAse I,

75ml DMEM

=100ml total, filtrate 0.45µm to make sterile

First, rinse your adhering cells once or twice with PBS, then add some of this buffer and scrape the cells off (room temp, so the the enzyme can do its job) let sit for a minute or so and resuspend CAREFULLY with a blue tip, then go through the cell strainer, avoid centifugation if you can. Hope this helps, good luck!!! The Heparin should provide additional anti-clumping activity, it may not be absolutely necessary, but since you have some really needy guys here, so I would probably give them maximum attention. GIve it a shot, I am optimistic!

Good luck!

Thomas :-)

So, I promised to follow up on this, giving you what we use for DNAseI treatment of cell suspensions (ahead of cell sorting). First of all, we order DNAseI powder (any quality can do, so it becomes more of a price related issue) from Sigma. We dissolve at 1 mg/ml in 0.15 M NaCl and 50% glycerol, divide into small aliquots and store at -20°C. To our samples we add 1/100th of a volume of this solution. Please note that DNAseI needs divalent cations, so any buffers containing chelators will interfere with it's activity !

HTH

Jan

1. Flow cytometry problem. As indicated by others, to prevent clumping, you should use Ca2+-free medium for flow cytometry. For example, you could you AutoMACS running buffer (Miltenyi Biotec), which is: PBS (NaCl, KCl, Na2HPO4, KH2PO4) + 2 mM EDTA + 0.5% BSA (pH 7.2). You can buy the buffer or make your own etc.

2. Resuspension of BM-derived macrophages problem. One way of avoiding scraping is to grow the cells in a 30 ml Teflon bag, e.g. PermaLife PL30. You place the bag in ice for 30 min, then you simply shake/massage the bag and the cells are readily resuspended - no cell trauma. You can then pour them into a 50 ml Falcon tube and mix with a 10 ml disposable plastic pipette. One caveat is that the cells may have another phenotype due top the Teflon surface; the cells tend to have elongated trailing ends (possibly due to downregulated RhoA activity).

In general, we do not really like cell suspension buffers with (high concentrations of) EDTA, or other chelators. They will compromise membrane integrity, for which bivalent cations are needed. This is confirmed by what we see in flow cytometry, just by looking at scatter distributions (FSC vs SSC; morphology). Cells from samples that were pre-purified by magnetic beads based procedures (e.g. Miltenyi; employing EDTA containing suspension buffers) don't look as nice as untreated cells. So, wherever possible, we try to avoid using buffers containing these kinds of constituents.

Macrophages tolerate short periods of 0Ca2+/EGTA (or EDTA) very well. The few times I used magnetic beads, I found that the cells did not look so well. I suspect that the magnetic bead separation (positive or negative) is more traumatic than short term exposure to Ca2+-free medium.

Just check the concentration of DPBS you are using. May be you need to optimize the concentration ( mostly by slight increase in the concentration).

I have not worked with BM derived macrophages but have cultured monocytes in conditions to generate macrophages and done extensive work with them on flow cytometry. First off, we found any scraping leads to considerable cell death as viewed by 7-AAD staining on flow. The best method we have found is to use TryplE for 15min at 37 degrees. Stop by adding RPMI+10% FBS and extensive pipetting with a 1ml pipet. Almost 90% of cells will detach with about 90% viability. We resuspend in FACS buffer and when checking for doublets with FSC-H vs FSC-A we have less than 5%. WE use only polypropylene tubes and have checked for loss of surface antigen due to use of TryplE and have found no problem for the markers we are using (CD80, CD163, CD14, CD16. CD206). Hope this helps. I would not recommend suspension cultures as that is an entirely different system with different expression of markers due to their non-adherent state.

Another solution widely used in flow cytometry is cell filters. I used it on lymphocytes that tend to clump. You pass your cells through the filter just before FACS analysis to remove any clumps of cells.

Absolutely right ! But, if you did not treat by DNaseI before, you risk loosing heaps of cells. So, treatment by DNaseI, combined with filtering just before running the sample (and definitely following the last centrifugation/resuspension).

I use rubber policeman to scrap off the cells in cold PBS, then use blue tip pipet up and down to make single cell suspension. After finished my staining, I push 500ul of cells through FACS tube with strainer (Falcon 352235). I have compared those cells with cold PBS, trypsine/EDTA lift cells, there was no difference in PI/Anexin V positivities.

In regards to clumping of bone marrow derived macrophages, I suggest you either put the cells through a syringe (22G) or use a 100 um cell strainer. I do this method as soon as I isolate the cells from the bone. This has been very helpful for me especially for immediate FACS analysis.

Also, you can do a series of sub gating to exclude doublets. I would first do a P1 gating for your population of interest. Then sub gate off of P1 for FSC-W and FSC-H. This should be a small gate (P2). Next off of P2 add another gate P3, which is SSC-W and SSC-H. After you do these series of gating, you should have discriminated against doublets. All subsequent gating should be off of P3 for you experiments.

Hi,

I am wondering whether you did digest macrophages from bone marrow ? Clumping of bone marrow-derived macrophages may be due to digestion procedure.

Best regards & have a nice weekend all of you !

Andrea

Dear Steven,

40µm is pretty large to get single cells. In case you would like to improve this step we provide cells strainer (pluriStrainer) with mesh sizes of 6µm, 10µm, 15µm, 20µm and 27µm. Here you chance to receive a real single cell solution is a lot better. Additionally we provide a Connectoring that fits to any 50ml reaction tubes. That you can use to support the flow through the strainer with low pressure (via a syringe) and avoid too much cell los. In case you would like to have more information just contact me or check our website: http://pluriselect.com/product-details/product/pluristrainer.html. I hope you will find the information helpful.

Best wishes

Michael

I found out that using trypsin to detach macropages is not affecting too much surface staining, However, if you prefer to collect them with the cell disociation buffer, I would try by resuspending the macrophages in PBS+0,5%BSA+2mMEDTA. This solution should keep them from clustering. It is the buffer used in the MACS separation protocol, in which it is necessary that cells are not clustered to avoid selection of the wrong cell type.

macrophages do not adhere to glass. They can be transfered to glass flasks or petri dishes. and use glass pipettes

I have worked with macrophages and do it exactly like Robert Landis (answered above). The only other difference is that I keep 2mM EDTA in my FACS buffer to keep cells from clumping during the run.

I find that macrophages adhere quite quickly in NON TC TREATED plates. By 24 hrs you can already remove non adherent debris and replace the growth media. I think debris from other cells in the BM tends to make them stick more vigorously to the plate. On removal,, gently rinse plate with RT PBS to get rid of all growth media, serum etc. Aspirate and then add 4ml (10cm dish) of ice cold PBS +EDTA, incubate for 5 mins then use a 5ml pipette to gently rinse them off the plate.

Using a PBS buffer with ETDA and BSA or FBS is the best in my hands. Also if you used bacterial petri dishes rather than tissue culture plates. Macrophages tend to adhere strongly to TC plates rather than Bacterial plates.

Good luck.

Hi Steven,

Lots of excellent advice here. In my humble opinion, you need to use EDTA and DNAse to prevent the clumping. And in the case of these DC cells DO NOT use a 40um strainer. Whilst theoretically that should help with single cell suspensions, in practice with cells with the morphology of DC, you are going to lose 80 % of your cells as you are. You can ensure single cell supensions by repeated pipetting (which can also detrimentally affect the cells) or use 100um strainers. If you have clumps, a good move is to let them settle by gravity, then remove the rest of the supension

Best wishes & good luck!

I've see Accutase used as gentle way of detaching cells when EDTA had too much of an activating effect already and you need as much immature macrophages as possible. Otherwise yes, PBS and EDTA is the cheapest version.

Don't add FBS to your PBS though as recommended here, it will bring so much magnesium ions in unknown/varying concentration that the action of your PBS/EDTA will be unreliable. Better titrate the EDTA to the level you really need. (EDTA acts by chelating the bivalent ions and thus interferes with the extracellular matrix binding proteins that cause the attachment.)

Btw here is more suggestions for special coated plates: https://www.researchgate.net/post/What_is_the_best_way_to_harvest_adherent_macrophages_out_of_48-well_plates