Frequently Asked Questions
If your question is not answered on this page, please do not hesitate to e-mail us with your question.
"In vitro" depletion of macrophages:
- (#13) Is it possible to use clodronate liposomes for depletion of macrophages "in vitro"?
Yes, it is possible to deplete macrophages in vitro, but please keep in mind that the method is specifically suitable for "in vivo" research. The reason is that clodronate released from dead macrophages or released in the culture medium by leakage from liposomes (dependent on the composition of the medium) cannot escape from the medium whereas clodronate, once released "in vivo", has a very short half life due to its rapid removal by the kidneys. Free clodronate molecules will not easily enter into cells because of their difficult passage of cell membranes (as well as liposomal phospholipid bilayers). However if they remain in the surrounding medium they may slowly accumulate into cells. See also reference: Claassen, I., Van Rooijen, N., and Claassen, E., 1990. A new method for removal of mononuclear phagocytes from heterogenous cell populations 'in vitro', using the liposome-mediated macrophage 'suicide' technique. J. Immunol. Meth.. vol. 134. pp. 153-161. (#32).
alveolar macrophages (AM):
- (#17) What about the administration route for clodronate liposomes in order to deplete alveolar macrophages (AM)?
Liposomes can be administered intranasally as well as intratracheally. The former administration route has been used several times (see literature in our website e.g. :
DeHaan, A., Groen, G., Prop, J., Van Rooijen, N., Wilschut, J. 1996. Mucosal immunoadjuvant activity of liposomes: Role of alveolar macrophages. Immunology, 89; 488-493)
The difference may be that all liposomes once administered intratracheally, arrive in the lung (alveoli), whereas liposomes administered intranasally (if administration is not well performed) may be spoiled via the oesophagos.
Animals die after injection of clodronate liposomes:
- (#4) My animals die during or immediately after intravenous injection of clodronate liposomes
- This may be caused by injection of a non-homogeneous suspension of the liposomes.
- The suspension of liposomes should be shaken or stirred before injection in order to get a homogeneous suspension. Liposomes will precipitate after some time. If injection takes too much time, they may even precipitate in the syringe If more animals are injected using the same syringe, this may also cause a differential dosing since the first animal will get much more (partly precipitated) liposomes than the last one.
- Another reason may be a too fast injection. If liposomes are taken from the refrigerator they should get the time to reach room temperature at least.
- (#5) My animals die some days after injection of clodronate liposomes
This may be caused by microbial contamination and/or activation of the animals shortly before or during injection. Please keep in mind that the absence of macrophages may cause an increase in e.g. virus titers, bacteria or yeasts.
It is also important to know that animals injected intraperitoneally with clodronate liposomes should be clean where injected. Please shave a small area of skin and treat with some alcohol before injection, since microorganisms may go in the animal together with the needle if it passes. Given that macrophages will meet a lot of liposomes, they may no longer be able to kill the microorganisms.
Application of a label to liposomes:
- (#12) Labeling Liposomes
Staining of liposomes to follow their fate in vivo.
It is important to keep in mind that a label used to mark liposomes should stick to these liposomes under all conditions. The consequence is that such a label should either be extremely hydrophilic in order to become firmly and exclusively associated with the aqueous phase of the liposomes OR extremely lipophilic (hydrophobic) in order to become firmly and exclusively associated with the lipid phase. Another possibility is to couple the marker covalently to one of the phospholipid molecules used to prepare the liposomes.
Most dyes are to some extent lipophilic, but liposomes will lose the dye during washing procedures, or as soon as they are injected in an animal in vivo. This is because the balance between the lipid phase associated dye and the aqueous phase associated dye is disturbed under these circumstances (see e.g. Van Rooijen, N., Van Nieuwmegen, R. 1978. Fluorochrome staining of multilamellar liposomes. Stain Technol. 53; 307-310).
Control liposomes may be labelled, e.g. with the fluorochrome DiI, since they do not affect macrophages. As a result the label will show the distribution pattern of the liposomes within tissues and their uptake by macrophages. We do not recommend to use DiI labelled clodronate liposomes for the following reasons: Clodronate liposomes will kill the macrophages; as a consequence, the DiI label will be redistrubuted as soon as the macrophages are dying and the label does no longer represent the actual distribution of the liposomes. So, liposomes should either contain clodronate to deplete macrophages or DiI to show which macrophages are taking up the liposomes (or whether the liposomes are able to reach the macrophages to be studied). Combination may lead to misinterpretation.
PROTOCOL for LABELLING LIPOSOMES with DiI:
- PBS: 0,012M phosphate; 0,8% NaCl, pH 7,4
- DiI solution:
Add 1 ml ethanol 100% to 2,5 mg DiI (Molecular Probes article number D-3911)
Sonicate for 1 min. in waterbath at 35 kHz (cristals should be dissolved)
- Add 10 microliter DiI solution per ml liposome suspension
- Shake liposome suspension thoroughly
- Incubate 10 min. at room temp. (dark)
- Centrifugate liposomes at 20.000 xG for 10 min.
- Remove supernatant
- Add sterile PBS and resuspend
- Centrifugate liposomes at 20.000 xG for 10 min.
- Add sterile PBS to original volume
- Store labeled liposomes dark at 4 degrees Celsius
ED.1+ cells and ED.2+ cells in the rat:
- (#6) ED 1+ cells in my rat spleen/liver did not disappear completely after intravenous injection of clodronate liposomes
- A1. Mature macrophages in the rat are positive for ED 1 and ED 2. However also their precursors which show no phagocytosis (directly after leaving the bone marrow) or little phagocytosis (shortly before their diffentiation into mature macrophages) are positive for ED.1.These precursors are negative for ED.2. As a consequence, ED 2+ cells will be depleted completely, but ED 1+ cells only partly. The percentage of ED1+ depletion depends on the actual ratio of precursors/mature macrophages and may be changed at later time intervals due to recruitment of new precursors.
Failure of activity:
- (#23) The clodronate liposomes received, did not work as expected.
Please be aware that both clodronate liposomes and control liposomes shipped to you are taken from large batches. These mother batches get a unique code and are checked for clodronate contents and possible contamination after preparation. Samples from these batches are sent to several (ca 30) different labs all over the world. As a consequence, if we get an email reporting poblems related to inactivity of the liposomes or contamination, we will check whether there are any other complaints from labs that did receive samples from the same batch as those sent to you. If not we will of course inform you as soon as possible future complaints will reach us.
Specific geographically related reasons for their failure might be caused by extreme temperatures during transport or storage. Liposomes should never be frozen or heated above 30 degrees Celcius at any time during transport or storage.
Moreover administration routes should be chosen in such a way that liposomes have an unhindered access to the phagocytic cells to be depleted (see information chapter). Please keep in mind that liposomes are particles and will not cross the endothelia of normal capillary vessels.
Also, specific markers for macrophages are required to establish their depletion from the tissues. The use of non-specific markers may affect the reliability of results.
Finally you should be sure the injection was performed in the right way. If e.g. after intravenous injection in the tail vein of mice, you will feel a resistance when applying the syringe and/or a bulb is formed next to the injection site, You did very probably miss the vein with the needle. Please ask a colleague with experience.
Intraperitoneal injection should be in the peritoneal cavity itself and not e.g. in the intestines. In order to prevent this, the skin should be shaved on the place where injection is planned, cleaned with alcohol and lifted in order to get some space between skin and the underlying organs.
- (#25) How to distinguish the tube containing clodronate liposomes from that containing control liposomes if labels are missing or seem to be wrong?
Is there any remaining volume of both tubes? In that case could you please place a similar volume of both tubes in the centrifuge and have a look to compare the pellets formed (1500 rpm, 5 minutes). The clodronate liposomes will have a different size pellet than the PBS liposomes.
The clodronate liposomes will form a pellet of about 1/3 of the volume, whereas the PBS liposomes will form a pellet of about 1/10 of the volume.
If both tubes contain the same size pellet, we are very sorry for the inconvenience and will send you new liposomes as soon as possible.
General directions for storage, treatment & administration:
- (#28) General directions for storage, treatment & administration.
*The suspension of clodronate liposomes, as well as that of control liposomes should be stored between 4 and 8 degrees Celcius. If storage lasts for only one day, liposomes may be stored at Room Temp.
*NEVER FREEZE the suspension since phospholipid bilayers may be disturbed and leakage of clodronate may occur!
*A temperature of over 40 degrees Celcius may also damage liposomes.
Directions for use:
*The suspension of clodronate liposomes as well as that of control liposomes should be used as they are upon arrival. However, since liposomes are particles, they will tend to precipitate. So before administration to animals, the suspension should be shaken in order to resuspend a possible pellet if formed at the bottom of the tube or on top of the suspension. A homogeneous suspension is required in order to warrant an equal concentration of liposomes per ml of it. If dilution of the suspension is required, please use PBS (phosphate buffered saline) or saline.
• For administration routes see: www.ClodronateLiposomes.org Home page menu> Information > Injection scheme.
• For intravenous injection, ca 0.1 ml of the suspension should be injected per 10 grams of animal weight. I.e ca.0.2 ml for a young adult mouse of 20 grams. Depletion of liver and splenic macrophages is complete after ca 24 hrs.
• For intraperitoneal injection allow ca 3 days for depletion, since liposomes have to reach the circulation by lymph flow via “ductus thoracicus”
• The final preparation is containing ca 5 milligrammes of Clodronate per ml of the suspension.
• Liposomes are sized at max. 3 microns. As a result their dimensions vary between 150 nanometer and 3 microns.
* Liposomes can be stored at 4 degrees C for at least 6 weeks.
Actually their activity lasts for much longer periods, since some scientists told me that the liposomal activity was still OK after half a year or more.
Our own measurements on loss of activity were not extended beyond 2 months. No leakage of clodronate was detected during that period.
However given that (clodronate) liposomes cannot be sterilized, neither by heating nor by irradiation, since such treatments could disturb the integrity of the phospholipid bilayers of the liposomes, we do not recommend to use them for longer periods of time. If any microbial contamination will occur, this could induce problems especially in macrophage depleted animals. Macrophages form the first line of defense against such a contamination.
• For specific macrophage subpopulations, please read the relevant literature. Publications can be found by searching REFERENCES, section: PUBLICATIONS.
• Please read the entire INFORMATION section and specific questions at FAQ.
- (#27) What about advantages and disadvantages of liposome mediated depletion of macrophages
Specific ADVANTAGES of the liposome mediated macrophage “suicide” approach:
* Applicable to macrophages in all vertebrate species. This is because specificity is based on functional activity of macrophages (phagocytosis) in stead of their antibody-mediated targeting.
*Consisting of biologically inert compounds (phosphatidylcholine & clodronate) that have already been introduced in the clinic (though separately).
*Compounds are inactivated after action; clodronate released from killed macrophages will rapidly leave the circulation in the kidneys and is secreted via the urine (half life ca 15 mins).
phosphatidylcholine is a normal component of cell membranes.
*No countereffects such as activation of non-depleted macrophages see: Van Rooijen, N. and Sanders, A. 1997. Elimination, blocking and activation of macrophages: three of a kind? J. Leuk. Biol. 62; 702-709.
*Specific depletion of tissue- or organ-specific macrophage subpopulations can be achieved by choosing the right administration routes.
Unevitable DISADVANTAGES of macrophage depletion:
*Since macrophages are influencing many other (non-phagocytic) cells by remote control (e.g. via cytokines and chemokines), long-term macrophage depletion may finally lead to cancelling of activities of non-phagocytic cells.
*Upon killing of macrophages, their remnants may circulate for some time.
How to handle Clodronate liposomes and control liposomes:
- (#20) How to handle the Clodronate liposomes upon arrival
The suspension of Clodronate liposomes as well as that of control liposomes should be used as they are, without dilution or any other treatment. They can be stored at 4 degrees Celcius when not used within a few days upon arrival. NEVER FREEZE LIPOSOMES!!
Liposomes (as particles) will tend to precipitate. For that reason the suspensions should be gently shaken before dividing these in smaller volumes or before injection in animals, in order to get homogeneous suspensions again and an even distribution of liposomes over the entire suspension. If stored at 4 degrees Celcius, liposomes should get the opportunity to reach Room Temperature before injection. NEVER INJECT COLD LIPOSOMES!!
- (#24) Is it possible to sterilize Clodronate Liposomes?
Sterilization of liposomes is difficult. First of all, if heated to a temperature required for sterilization, the liposomal structure will be destroyed. Also sterilization by radiation may destroy their phospholipid bilayers. In a certain expt in which clodronate liposomes were used in humans (within the synovium of knee joints: ref: Barrera, P., Blom, A., Van Lent, P.L.E.M., Van Bloois, L., Storm, G., Beijnen, J., Van Rooijen, N., Van De Putte, L.B.A., Van Den Berg, W.B. 2000. Synovial macrophage depletion with clodronate containing liposomes in rheumatoid arthritis. Arthritis. Rheum. 43; 1951-1959.), small unilamellar liposomes were produced that could pass filters not allowing the passage of bacteria. However such liposomes cannot be produced at a regular basis by us. Nevertheless liposomes are prepared under very clean conditions. Also the possible danger of contamination during injection seems to be a larger problem. If animals are affected this will be in most cases the result of microorganisms that are already present, but get a change to affect the animals as a result of the absence of macrophages.
Increasing the clodronate/liposomes concentration:
- (#9) Is it possible to raise the clodronate concentration within the liposomes?
- No, the concentration of clodronate in the aqueous compartments within the liposomes is limited by the solubility of clodronate and is kept at a maximum in the standard clodronate-liposomes suspension.
- (#10) Is it possible to raise the injected dose by enhancing the number of liposomes per ml suspension?
- This cannot be recommended for intravenous injection since the fluidity will be decreased and this may lead to blocking of capillaries. However it may be tried e.g. for local injection in loosely woven tissues or for intraperitoneal injection from where the liposomes are filtered before they gradually reach the circulation.
- (#11) Can the injected volume of the clodronate liposomes suspension be raised?
- Intravenous injection should not be more than 0.1 ml per 10 grams of body weight. However for intraperitoneal injection the injected volume may be increased considerably. For subcutaneous injection the maximum volume to be injected depends on the storage capacity of the injection site.
- (#15) Is it possible to compare the effects of free (non-encapsulated) clodronate with those of liposome encapsulated clodronate in vivo?
Since the half life of clodronate in vivo is in the order of 15 minutes, there is quite a difference between injection of a high dose of free clodronate at once, which will then be reduced to half of its original value each 15 minutes AND injection of the same dose in liposomes which will be released from dead macrophages slowly.
In the first case there is a high concentration that will be reduced rapidly. In the second case there is a much lower concentration that will be maintained for a longer period of time.
Intraperitoneal & intravenous injection: The difference:
- (#26) What about the administration route for depletion of spleen and liver macrophages
Intravenously injected clodronate liposomes induce depletion of macrophages in spleen and liver within 24 hours (see useful references in website).
Intraperitoneally injected liposomes take more time before a substantial depletion is reached in liver and spleen, but contrary to intravenous injection, by this route peritoneal macrophages are also depleted
Liposomes have to be carried from the peritoneal cavity to the circulation by the lymph flow, which is - contrary to the blood flow - a passive form of transport. The implication is that intraperitoneally administered liposomes reach the spleen and the liver after a longer time than intravenously injected liposomes. Also they are arriving more gradually and over a longer period of time.
Reference: Biewenga,J., Van der Ende,.B., Krist,L.F.G., Borst,A., Ghufron,M. and Van Rooijen,N. 1995. Macrophage depletion and repopulation in the rat after i.p.administration of Cl2MBP- liposomes: Depletion kinetics and accelerated repopulation of peritoneal and omental macrophages by administration of Freund’s adjuvant. Cell Tissue Res. 280; 189-196.
In mice e.g.: Fraser,C.C., Chen,B.P., Webb,S., Van Rooijen,N. & Kraal,G. 1995. Circulation of human hemopoietic cells in Severe Combined Immunodeficient mice following Cl2MDP-liposome mediated macrophage depletion. Blood, 86; 183-192.
In general, ca 0.1 ml of the suspension of clodronate liposomes per 10 gram of animal tissue should be injected for depletion of splenic macrophages. For depletion of liver macrophages (Kupffer cells) half of that dose may be used.
In case of intraperitoneal injection with the intention to deplete splenic and liver macrophages a first injection on day 0 may be followed by a second injection on day 3 (see e.g. Biewenga et al. 1995)
Long term depletion of macrophages:
- (#8) Is it possible to maintain macrophage depletion for longer periods of time?
- Under normal conditions i.e. in the absence of activation by some microbial products, macrophage populations such as Kupffer cells in the liver and red pulp macrophages in the spleen start to repopulate their compartments after ca. one week.
- Other macrophage populations such as marginal metallophilic macrophages and marginal zone macrophages in the spleen (after intravenous injection) and macrophages in lymph nodes (after subcutaneous injection in their draining areas) remain absent for much longer periods of time (see relevant literature under manuscripts).
- In order to achieve a prolonged macrophage depletion in SCID mice, Fraser et al (Blood, 86; 183-192, 1995) tried several injection schedules in which one undiluted injection of clodronate liposomes was followed by subsequent diluted doses of clodronate liposomes each 5-7 days.
- However one should keep in mind that macrophages regulate functional aspects of various non-phagocytic cells. As a consequence, their long term absence may ultimately lead to the functional inactivity or even the disappearance of these non-phagocytic cells.
Macrophage precursors (monocytes):
- (#7) Is it possible to deplete macrophage precursors?
Depletion of monocytes and indirect depletion of Macrophages
For a long time, the generally accepted theory was that monocytes, as precursors of mature macrophages and dendritic cells (DC) were produced in the bone marrow and released in the circulation. After a short period (2-3 days) in the circulation, they left the circulation by extravasation and entered the parenchyma of tissues (organs). There they did finally maturate into highly phagocytic resident macrophages, inflammatory macrophages (in case of an inflammatory area) or antigen presenting dendritic cells.
As a consequence, the finding that a single intravenous injection with clodronate liposomes did only deplete macrophages in organs where liposomes had an unhindered access to resident macrophages as in liver and spleen (in case of intravenous injection) was not surprising. Neither two or three injections, repeated with an interval of one week or more, appeared to be sufficient to deplete macrophages from organs where vascular endothelia did form a natural barrier between the circulation and the parenchyma of such tissues (early unpublished results).
To our surprise however, some collaborating investigators, who insisted to try the depletion of macrophages in such organs, in spite of our contra-recommendations, appeared to be successful in macrophage depletion after several injections, repeated after shorter intervals.
This could later be explained by the results of a crucial study on subpopulations of mouse blood monocytes in the circulation, their maturation and their kinetics after depletion by clodronate liposomes (Sunderkotter et al. 2004).
These authors found that virtually all peripheral blood monocytes were depleted at 24 hrs after intravenous injection of clodronate liposomes. The first cells reappearing at two days after injection of clodronate liposomes were BM-like Ly-6chigh monocytes, whereas
Ly-6clow monocytes were detected in the circulation from 7 days after depletion.
Ly-6chigh monocytes that reappeared on day 2 after depletion were pulse-labeled in vivo by injecting DiI labeled fluorescent liposomes without clodronate. By 3 days after the DiI labeling, Ly-6chigh monocytes had developed into Ly-6clow monocytes, whereas the vast majority of unlabeled monocytes at that time were again of the Ly-6chigh BM type.
These studies clearly demonstrated the rapid kinetics of monocytes in the circulation. Moreover, under inflammatory conditions, a significant increase in immature Ly-6chigh monocytes was observed (Sunderkotter et al. 2004).
So, depletion of monocytes from the circulation by injections of clodronate liposomes, repeated at shorter time intervals (e..g. two days), are needed for an effective depletion of mature macrophages and dendritic cells in organs where liposomes have no direct access to mature macrophages. Interestingly, this treatment caused a delayed disappearance (7-21 days post injection) of macrophages and dendritic cells from the endocrine pancreas at a time when monocytes, macrophages and dendritic cells had already repopulated the circulation and spleen. The depletion of macrophages and dendritic cells from the endocrine pancreas was accompanied by a total disappearance of lymphocytes from the pancreas. All depleted cells started to reappear in pancreatic inflammatory infiltrates from 28 days onwards. (Nikolic et al. 2005). Obviously, mature macrophages and dendritic cells are dependent on monocytes for their reappearance and lymphocytes in turn depend on cells of the monocyte/macrophage lineage for their entrance in both pancreas and brain (Tran et al. 1998).
For complete references see our list of publications (references)