The Biofluid assay facility performs clinical chemistry and hormones/cytokines/chemokines profiling in a variety of biological samples (mouse, rat and human) including plasma/serum, urine and conditioned media from cell culture or explants experiments.
- Advising on experimental design (see also Resources below)
- Clinical Chemistry using the Cobas C111 robot
- Multiplex Hormones & Cytokine assays (Luminex technology)
- Blood / urine collection: see Resources here below
General terms apply as described here.
Biofluid assay equipment are exclusively used by the facility staff. (see Resources here below).
Hazardous samples containing pathological bacteria, viruses or radioactivity will not be accepted.
The nature and quality of samples has a major impact on some assays. Users therefore need to contact Gilles.Willemin@unil.ch before collecting samples in order to discuss the sample collection procedure.
|Gilles Willeminemail@example.com||2022||021 692 39 57|
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Rooms, equipment & link to other relevant facilities
Protocols, reagents & reference values
Blood sampling in mice: Experimental Design
It is always advised to contact us before blood collection to define the optimal conditions for the analysis of interest.
The following sections provide information and recommendations to users regarding blood collection. The nature of the required sample (serum vs plasma, etc) is detailed in the Clinical Chemistry and Hormones & Cyokines pages.
The Biofluid assay facility can also help users for blood collection (please contact us).
Blood Collection Procedures
Currently approved blood collection schemes are:
- Non terminal bleeds
- Submandibular vein puncture, in awake or anesthetized mice (large volumes, replaces the retroorbital puncture technique);
- Saphenous vein puncture, in awake or anesthetized mice (for repeated, small bleeds);
- Tail vein puncture (convenient for repeated, very small bleeds, or single small bleed)
- Terminal bleeds
- Retroorbital blood puncture, in anesthetized mice (large volumes)
- Intracardiac blood puncture, in anesthetized mice (large volumes)
The maximum authorized non-terminal bleed volume is 1.4 % of the body weight per period of 14 days (20 % of the blood volume, which is 7-8 % of the body weight) = 0.28mL blood for a 20g mouse and 0.42mL / 30g.
Nature of samples
The blood collection conditions will depend on the type of biological sample required for the analyses. Please contact Gilles Willemin for details of the blood sampling procedure before collecting the samples.
Serum. Collect blood into tubes (with/out coagulation activators). Incubate at room temperature for 15 minutes (clotting). Centrifuge 10’ at 4 degrees, 4,500 rpm. Transfer supernatant into Eppendorf tubes of 1.5 ml preferentially, or 0.5ml (no strips).
Plasma. Collect blood into tubes with the appropriate anticoagulant at room temperature. Centrifuge 10’ at 4 degrees, 4,500 rpm. Transfer supernatant into Eppendorf tubes of 1.5 ml preferentially, or 0.5ml (no strips).
Store samples for Luminex at -80°C and samples for clinical chemistry at -20°C or -80°C.
Sample labeling. Tubes should be clearly labeled with easy codes (e.g. numbers #1-100) using permanent markers. Please include a list with detailed information about the nature and the number of samples.
Strictly avoid hemolysis. Hemolysis can have a moderate to huge impact on the outcome of some specific tests. Ways to avoid it include:
- Never shake or whip blood samples (inversion is ok)
- Never draw/eject too quickly blood from a capillary, needle, tip or syringe
- During tail bleeding, do not apply too much pressure on the tail.
Parameters to be controlled include
- Chronobiology: as the metabolic status is highly dependent on the circadian behaviours (feeding/fasting, activity/sleep, etc), blood collection should be performed always at the same time of the day, over a limited period of time (a couple of hours), and genotypes should always be randomized (see below).
- Stress: All aspects of experimenter-induced stress are expected to significantly and/or durably alter blood parameters in mice. Therefore, special attention should be paid to drastically reduce the time of exposure to the following stresses, and/or reduce their impact using appropriate acclimation procedures defined specifically for each physiological parameter of interest. Other practical aspects to consider in the design include the configuration and proximity of housing and experimentation rooms, etc.
- Transportation: While transportations from one facility to another have well-known, widespread physiological effects that require an acclimation/recovery period of at least one week for restoration of homeostasis, short transportations from a housing room to an experimentation room similarly induce stress-related alterations of homeostasis (http://ilarjournal.oxfordjournals.org/content/47/4/364.full.pdf+html ; http://lan.sagepub.com/content/29/2/132.full.pdf). Therefore, optimal transportation/acclimation procedures should be defined specifically for each study and parameter measured.
- Manipulation: Even short manipulation times in the one-minute range are long enough for the expression of stress-induced alteration of some sensitive parameters. Thus, manipulation time should be as short as possible, and specific manipulation / acclimation procedures should be set specifically for each parameter of interest. During acclimation procedures, only a careful and respectful manipulation will elicit a positive learning curve and reduce the manipulation stress over time.
- The missing cage mate effect: Removing mice from a cage will stress remaining cage mates in a time - and sequence - dependent manner, so that the last mouse will be subject to the highest stress. Thus, mice should be either isolated one per cage before the blood collection (with an appropriate acclimation period), or the time between blood collection in the first and last cage mates should be as short as possible, and randomized for genotypes (see below).
- Inter-individual stress transmission: Mice are stressed by the smell of blood and by vocalisations of other congeners present in the room. Thus, last mice manipulated in an experiment are exposed to more environmental stress than the first ones. Therefore, numbers of mice per experiment should be kept within a reasonable range and genotypes should be randomized (see below).
- Randomization of mice during the blood collection: Key to any experimental design, randomization of genotypes should be carefully applied to take into account all potential sources of artefacts related to chronobiology and stress during blood collection sessions. As best practice, all genotypes should be randomized in each cage, and bleeds completed for all individuals in a cage before processing to the next one. Should two diet treatments (A & B) be compared, cages with diet A, then B should be alternated. Same principle can be applied to genders, should they be compared in the same experiment day.
- Proper blood collection technique is of utmost importance to reduce the manipulation time and stress as well as the recovery time. The MEF staff can perform, or teach, some of the bleeding procedures.