FAQ

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
  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 usually do not send 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:
  Materials:
  - 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)
  
  Method
  - 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.



• (#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.
  

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 serilize 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.
  

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?
  - Whereas intravenously injected clodronate liposomes have no access to mature macrophages in e.g. the gut, due to the fact that liposomes are not able to cross the vascular endothelium of capillaries, Galeazzi et al. (Am J. Physiol. 278 G259-G265, 2000, see also under manuscripts) described the effects of intravenously injected clodronate liposomes on the inflammation induced recruitment and infiltration of F4/80 positive cells into the jejunum of mice. With respect to this question, the following is important: Precursors of macrophages (monocytes) recruited to e.g. inflammatory areas seem to be depleted by clodronate liposomes in the circulation. In such cases, several injections with clodronate liposomes have to be given shortly after each other, since 1. liposomes do not survive for long in the circulation and 2. new monocytes can be recruited within a relatively (compared to mature macrophages) short time. In the studies of Galeazzi et al., clodronate liposomes were given 4 hrs. before, and 1,2 & 4 days after the infectious agent to warrant depletion of recruited precursors as much as possible. - Obviously they got a substantial and in their case also a sufficient depletion, but a nearly complete depletion has never been reported. - There are many more studies in which the described effects of clodronate liposomes can only be explained by assuming an effect on macrophage precursors either in the circulation or at the level of monocyte/endothelium interaction. With respect to the Blood Brain Barrier (BBB), the latter effect may be mediated by the incorporation of mannose residues in the clodronate containing liposomes as was the case in the studies of Huitinga et al. (J. Exp. Med. 172; 1025-1033, 1990), describing the effects of mannosylated clodronate liposomes in an Experimental Allergic Encephalomyelitis (EAE) model. - However in studies where clodronate liposomes were repeatedly (2 times per week) and intravenously given to normal mice, mature macrophages in various organs where vascular barriers prevent a direct access to mature macrophages, were not depleted even after one month, emphasizing that monocytes recruited to maintain the normal turnover of macrophages in these organs, are not substantially affected.
  Please see also the NEWS SECTION:
  Blood monocytes (the precursors of mature resident macrophages) can be depleted by intravenous injection of clodronate liposomes.
  In: J. Immunol. 2004 Apr 1;172(7):4410-7.
  Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response.
  by: Sunderkotter C, Nikolic T, Dillon MJ, Van Rooijen N, Stehling M, Drevets DA, Leenen PJ.