![]() This inherent package leakage is referred to as the Maximum Allowable Leakage Limit (MALL). Creating leaking samples of known leak sizes is more complex and requires a thorough understanding of package, product, and leak dynamics. Proper use of positive controls and knowledge of how alternative approaches are applied to challenge a test method support the validity and reliability of the method. Given that the experimental design has only two conditions, leaking and non-leaking, this variable is extremely important to understand. Conventional thought is that non-leaking samples (or assumed non-leaking samples) are simply sealed packages in which no leak should be present. Realistically, all packages leak to some degree within the requirements of that package application. In order to live up to the Facility's mission of assuring quality control and reproducibility, facility staff will not assist with running samples when the necessary compensation controls are not provided by the investigator.Package integrity can be defined as a package’s ability to prevent product loss, maintain product sterility, and in some cases, prevent oxygen ingress or maintain sub-atmosphere headspace pressures. The characterization of integrity separates packages into leaking and non-leaking samples and allows for the validation of a test method to be a very simple experiment. There are only two conditions, non-leaking and leaking. Samples representing both groups are subjected to a test and the results for each should be measurably different from the other. Samples with either a non-leaking circumstance or a known leak defect are introduced to the test and should be correctly identified as non-leaking or defective. The lack of proper compensation controls may yield misleading, confusing, and inaccurate data. Please Note: The facility's mission is to serve investigators in their quest to obtain accurate data. * any antibody that is compatible with the beads See Tandem Fluorochrome Conjugated Antibody Best Practices There are exceptions to these rules when using tandem fluorochromes. tube 4) anti-CD8 APC stained splenocytes (or antibody against some other high density antigent).tube 3) anti-CD8 PerCP stained splenocytes (or antibody against some other high density antigent).tube 3) anti-CD8 PE stained splenocytes (or antibody against some other high density antigent).tube 2) anti-CD8 FITC stained splenocytes (or antibody against some other high density antigent).Because of the necessity to have brightly stained cells at a relatively high frequency (i.e, above 10% of the population) for accurate compensation, it may be necessary to use the same antibody that stains the high density antigen while varying the fluorchrome for each tube (see the following example), except when using tandem fluorochromes (see following section about using tandem fluorochromes).Įxample: For mouse splenocytes stained with FITC, PE, PerCP, and APC conjugated antibodies, compensation controls should include: If beads are not used, then cells expressing high levels of antigen (does not have to be an antigen of interest in the experiment) are stained with a fluorochrome-conjugated antibody that yields brightly stained cells. For experiments that cannot spare cells for compensation, do not have enough positive events, or have only low antigen expression, compensation beads are recommended. The beads are stained as if they were cells using the same antibodies and fluorochromes that are used in the experiment, producing both a negative and bright positive population for each color. Several vendors sell beads specifically for use as compensation controls. It is important that each compensation tube have a population of brightly stained cells (or beads) in order for the spill-over values to be accurately determined. Each of the compensation tubes is subsequently run to establish the spill-over values of each fluorochrome into the other fluorescent channels. The negative control (unstained cells) establishes the background fluorescence of the experimental samples and is used to set the baseline PMT (photomultiplier tube) voltages of the instrument. The proper compensation controls include a negative control (unstained cells are recommended) and one tube each of cells (or beads) stained positively with each of the fluorochromes used in the experiment. Compensation controls MUST match the exact experimental fluorochrome.Background fluorescence should be the same for the positive and negative control (e.g, positive cells vs negative cells, or positive beads vs negative beads).Controls need to be as bright or brighter than any sample the compensation will be applied to.(See for an in-depth explanation of compensation.) ![]() Spectral overlap between fluorochromes in multi-color experiments requires the use of fluorescence compensation controls.
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