Use normal precautions for handling animal products
General Description
Normal mouse serum is tested for complement activity and certified to possess functional classical and alternative pathways of activation. NMS was prepared from mixed gender CD1 mice. Each sample of blood was collected without anticoagulants and, after coagulation, the liquid portion was separated by centrifugation.
Serum was filtered through a 0.22 µm filter, aliqoted and frozen at -80℃. Physical Characteristics & Structure NMS is a clear, straw-colored liquid containing all proteins of normal mouse serum. Although the NMS is filtered through 0.22 µm sterile filters and is aliquoted into sterile containers, it is not packed under strictly sterile conditions and is therefore not certified as sterile.
Function
NMS is tested for classical pathway hemolytic activity using antibody-sensitized sheep erythrocytes and for alternative pathway function using rabbit erythrocytes. The Certificate of Analysis provided with each lot gives a description of the assays and specific titers for the serum. Assays Mouse complement is famously difficult to assay (Ref 1, 2 & 3). Mouse serum rapidly loses its complement activity and the typical titer of mouse serum is far below the 150-200 CH50 units/mL found with human serum. Mouse strains differ greatly in their complement CH50 titers (Ref 2, 4 & 5) and a great many common mouse strains are genetically deficient in C5 (Ref 6 & 7) meaning that they cannot lyse cells even if they have an otherwise fully functional complement system. The exact reasons for low titers in C5 sufficient strains are not entirely clear and they may be different for different mouse strains. However, a common reason is that mouse classical pathway components are not efficiently activated by the standard antibodies bound to EA. We have developed a unique hemolytic reagent (MCAR = Mouse Complement Assay Reagent) that sensitizes sheep erythrocytes to provide improved mouse CH50 titers. For example, normal mouse serum from CD1 mice exhibited a CH50 of only ~1 unit/mL in the assay system described below, however, with MCAR present the CH50 was ~8 units/mL . That is 8-fold higher complement titer with MCAR. At this time we cannot say how many mouse strains this reagent will work with, but we have used MCAR with three and it worked equally well with all.
Mouse serum is also extremely unstable outside of the mouse. We have observed a rapid loss of complement activity after thawing (100% loss if left at 4℃ overnight).
Thus, we advise rapidly thawing NMS and immediately using it in assays or if it is necessary aliquoting and freezing the NMS. It should be thawed rapidly in a water bath, moved immediately to wet ice when thawed, aliquoted and re-frozen as rapidly as possible. Upon use, thaw only when the experimental setup is ready for the NMS sample and keep it on wet ice after thawing.
In complement assays, every lot of mouse complement will have a different titer even from the same strain of mice. Preliminary assays will need to be done to determine the correct amount of NMS to use. Normal sensitized sheep cells (EA) work well if the lysis is boosted by MCAR. Most NMS samples generally contain a lot of hemoglobin and therefore the more serum used the higher the background. A sample assay protocol is shown in the table below where the total assay volume was kept to a minimum at 100 uL/assay. In general, tubes should be set up with buffers and MCAR at room temperature. Then EA (antibody-sensitized sheep erythrocytes) are added, mixed and incubated 5 min at room temperature. Do not centrifuge the EA after they have been mixed with MCAR and do not make a master mix of EA and MCAR and then pipette into the tubes as this will result in inconsistent results. EA are used at 5 x 108/ml in GVB⁺⁺ and all dilutions are done in GVB⁺⁺ buffer. After MCAR has incubated with the EA cells for 5 min or more at room temp the NMS is added to start the assay. The tubes should be mixed and moved to a 37oC water bath for 30 min with occasional (every 5-10 min) resuspension by vortexing. After 30 min at 37℃, the assays are diluted with 200 uL cold GVBE, mixed by vortexing, centrifuged to pellet the unlysed cells and 200 uL of the supernatant should be transferred to a flat-bottomed microplate and the absorbance read at 415 nm.
Alternatively a microcuvette and spectrophotometer may be used. Percent lysis is calculated by subtracting the background A415 values (average of #1 and 2) from the A415 vales for each of the experimental assays (e.g. #7-22) and dividing by the maximum lysis (average of #5 and 6) minus background and then multiplying by 100.
Applications
NMS is used to provide a source of mouse complement for hemolytic assays. This NMS complement serum has been pre-tested and certified to exhibit fully functional classical and alternative pathway complement activation.
Precautions/Toxicity/Hazards
The source is mouse blood, therefore precautions appropriate for handling any animal blood-derived product must be used.
MSDS is available upon request.
Specifications & Purity
≥30.0 mg/ml，0.22 µm filtered
Bioactivity
≥1.0 CP50 Units/mL active classical pathway ≥40 AP50 Units/mL active alternative pathway
