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Frequently Asked Questions If your question is not answered on this page, please do not hesitate to e-mail us with your question.
20 question(s)
"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).
Account blocked:- (#19) Why is it impossible to log in / is my account blocked?
BLOCKING of ACCOUNT
Blocking of an account may have three different reasons:
1. We have sent you an invoice more than two months ago and did not receive payment nor any reaction, in spite of sending you two reminders afterwards. Blocking will be ended as soon as the invoice has been paid in full. Please keep in mind that we can only maintain a low contribution per shipment, if invoices are paid without problems. We have to spend your money for technical assistance and materials but do not want to spend it for secretarial assistance. On the other hand, I myself did not become a scientist in order to send reminders.
2. It came to our attention that you did not stick to the conditions for delivery of our clodronate liposomes. These conditions are agreed with, each time an order is placed via our website and are written in detail under the chapters COLLABORATION, COSTS & MANUSCRIPTS of our website.
3. Shipment of liposomes to your address was returned to us and marked: undelivered, incorrect address.
Please see: Van Rooijen, N. and Sanders, A. 1994. Liposome mediated depletion of macrophages: Mechanism of action, preparation of liposomes and applications. J. Immunol. Meth. 174; 83-93 or Van Rooijen, N., Van Kesteren-Hendrikx, E. 2003. "In vivo" depletion of macrophages by liposome-mediated "suicide". Meth. Enzymol. 373; 3-16. for preparation of your own clodronate liposomes .
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.
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
Contribution to costs:- (#14) Why do I have to contribute to the costs of production, packaging and shipment of clodronate-liposomes ?
Although terms and conditions for delivery of clodronate liposomes have been clearly described in our website and have actually to be agreed on for each individual shipment, You will find here the reasons why you are asked to contribute to the costs of the production and shipment of clodronate liposomes for collaborative research, in spite of our attempts to reduce these costs to a minimum:
**Since we developed the technique for depletion of macrophages with liposome-encapsulated clodronate, there has been a gradual but continuous increase in requests to receive such liposomes from our group. So, I needed a continuous supply of clodronate.
I could get this regular supply from Boehringer Mannheim (now Roche Diagnostics) under some conditions, one of these being that I was permitted to distribute their clodronate (that we encapsulate in liposomes here in our laboratory) only for collaborative studies in which I remain responsible for the appointments with the company (e.g. sending a manuscript to the company for their approval, which automatically follows if I do not hear from the company within 2/3 weeks).
**Apart from this, and in spite of the fact that clodronate is provided at extremely low costs by Roche and phospholipids are purchased cheaply in a large quantity at once, costs of production and shipment of liposomes were increasing and could no longer be paid for in full, especially not since my retirement from the Department of Molecular Cell Biology of the VUMC. Over the past years, I have informed (on the website) future collaborators who needed regular delivery of clodronate liposomes, that they should incorporate the costs for the liposome shipments in their required budgets before submitting their grants. However this did n't work. So, since mid 2001 we have made very clear statements on the website on costs and conditions. We do not send invoices to countries or research groups for which payment will be a serious obstacle, but this should be appointed before ordering. Costs are only a fraction of the real costs for personal (technician), production, packaging and shipment, since numerous facilities and materials are provided by the department.
**At moment you can purchase clodronate from Sigma (1 gram ca. Euro 105 , product D4434) and you can purchase the phosphatidyl-choline and cholesterol from Sigma too. For preparation of your own clodronate-liposomes using these reagents you can follow the detailed description of their preparation in the following reference:
Van Rooijen,N. and Sanders,A., 1994. Liposome mediated depletion of macrophages: Mechanism of action, preparation of liposomes and applications. J. Immunol. Meth. vol. 174. pp. 83-93.
OR (more recently):
Van Rooijen, N., Van Kesteren-Hendrikx, E., 2003. 'In vivo' depletion of macrophages by liposome-mediated 'suicide'.. Methods in Enzymology. vol. 373. pp. 3-16.
Please be aware that the foundation 'Clodronate Liposomes' is a non-profit organization. Its aim is to make clodronate-liposomes as well as knowledge on their application, available to research groups that do not have the required facilities. See also FAQ 16 & 18.
- (#21) What about payment of orders
Please do not pay before an invoice is sent (or see FAQ 22). This will normally be done after some orders or after some months. In general we will only send an invoice when Euro 150 or more is due. The implication is that no invoice will be sent if, in addition to the first shipment of a maximum of 25 ml (Euro 130), no subsequent shipments were ordered. All invoices (per email) should be paid within 3 months. If no payment has been received within 3 months, your account and any other new accounts from your institute will be blocked automatically. See for more information on the reasons for costs: section Information, FAQ, questions 14, 16 & 18. When ordering liposomes, costs and expected shipping date will automatically appear on the order form when clicking on it. This is to show you this information before the final order should be sent. You can follow your order history and payment history via “MY ACCOUNT” on the website. Please be aware that the foundation Clodronate Liposomes does not have a VAT registration.
- (#22) Payment without an invoice
If necessary for you to pay within a short time (e.g. before the end of the year), you can pay the balance of your account by wiring as indicated below in the "pro forma" invoice. In this case your NAME as well as your INSTITUTE and CITY should be clearly indicated with your payment, in order to prevent our research hereabout.
ClodronateLiposomes.org
John Johnson
University of Xxxxxx
Department of Yyyyyy
Address
City
Country & Postcode
Email address user
Invoice number
Invoice date
Total amount due on invoice date: € xxx
Order Date; Price (€); MLs Cl; MLs PBS; Shipping; Tracking nr
aaaa 3/12/2009 130 25 25 fedex 797417435997
bbbb 2/12/2009 130 25 25 fedex 797340887122
cccc 1/15/2009 130 25 25 fedex 797260634442
dddd 12/4/2008 130 25 25 fedex 799411071808
Please visit www.clodronateliposomes.org/myaccount/ to view your invoices, orders and payments.
Please note that payment is due within 30 days.
Please include the invoice number (xxxxxxx) and your name in your payment.
Accounts that are more than three months overdue will be suspended.
Stichting (Foundation)
Clodronate Liposomes
www.clodronateliposomes.org
Kruisweg 59
2011 LB Haarlem
The Netherlands
Chamber of Commerce 34173596
ABN AMRO Bank
Account: 62.88.00.738
SWIFT: ABNANL2A
IBAN: NL32ABNA0628800738
Bank address:
Apollolaan 171
1077 AS Amsterdam
The Netherlands
Please send checks to:
Dr. N. van Rooijen
Moleculaire Celbiologie
VUmc
Fac. der Geneeskunde
Van der Boechorststraat 7
1081 BT Amsterdam
The Netherlands
Registered checks:
Dr. N. van Rooijen
Moleculaire Celbiologie
VUmc
Fac. der Geneeskunde
P.O. Box 7057
1007 MB Amsterdam
The Netherlands
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.
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!!
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.
production & shipment schedules:- (#16) When should I order & when will liposomes arrive?
On monday morning (9.00 Hr AM, Amsterdam time) all orders to be shipped in any particular week, will be collected from the website-associated order list. Packages are usually prepared and shipped the day after.
Orders received after that time will be shipped the next week at the earliest. Although we usually prepare a little more of the liposome-suspensions than required to satisfy all requests, our weekly production schedule is based on those requests.
From March 1, 2008, all liposomes will be shipped by Fedex, since too many packages were disappearing or were returned to us because of an incorrect address. Please check your address before registration, if you are new in your laboratory, and instruct other people in your lab when you expect a package from us.
If you want to be sure that liposomes are present, when you need these in an ongoing experiment, you should order at least 2 weeks before. Still better is to start the experiment when the liposomes are already stored in your refrigerator. Although we usually ship weekly, illness or other unforeseen circumstances may interrupt this process!.
When your order did not arrive within ca 2 weeks there are 2 main possible reasons. 1. The package was refused by your organization because your name was not known to people distributing the mail internally or your address was incomplete. 2. Another person has accepted the package without mentioning this to you. If this happens more than one time, your account will be blocked untill everything has been solved, because too much time and materials are spoiled for these reasons.
- (#18) What is the maximum volume that I can order at once ?
!! 25 ml's of both the suspension of clodronate liposomes and the suspension of control liposomes (containing PBS only) can be ordered for shipment in the next week.
!! For orders of more than 25 ml's you will be asked for an explanation (experimental design) to be given on the order form.
!! Orders of more than 50 ml's in larger animals have to be discussed before in detail and their urgency needs to be clearly demonstrated and must be authorized by email. They will probably be refused, since these orders disturb our production schedule too much and may not go at the cost of experiments in mice and rats. If admitted by NvR we require a period of at least 3 weeks before shipment will take place and we will inform you about the costs of the order when we receive your request per email.
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