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Can I return this product? Antibodies with known specificity and known optimal staining condition should be used to test if the dissociation protocol preserves the cell subsets of interest. Antibodies that have been shown to specifically detect targets with other techniques such as western blot might not always work for flow cytometry-based generic 25c-4.

Therefore, new antibodies should always be tested for specificity prior to use. This should be done using a known positive control cell type, and a known negative control cell type rather than relying on an isotype control antibody. All new antibodies should be titrated to yield optimal concentration prior to use. The ideal concentration should allow maximal separation between the positive and the negative control cells, while minimizing signal of negative control cells i. Antibodies that detect surface antigens should be used during live surface immunostaining. Note that some antibodies that detect surface transmembrane antigens were created to detect the intracellular portion of the antigen i.

Antibodies that detect intracellular antigens should be tested for appropriate permeabilization conditions. Different permeabilization reagents might be required for different antibodies. Additionally, steps of permeabilization generic 25c-4 different reagents, if needed, should also be tested. Every antibody should be tested for their specificity and for the appropriate concentration prior to use for staining. This protocol is applicable to human tissues extracted by surgery or to animal tissues isolated after dissection. To preserve tissue viability, samples should be transported to the laboratory for further preparation as rapidly as possible.

The dissociation protocol presented here was tested on samples that were processed between 30 minutes and 4 hours after surgical resection Leelatian et al. Additionally, samples should be transported in sterile PBS, appropriate experimental medium, or other sterile transport medium that has been tested to preserve cell viability and representative cell subsets for specific tissue types. Samples should be entirely submerged in the transport medium in a closed container. As described in Basic Protocol 1, tissue should be mechanically dissociated into fine pieces 1—3 mm generic 25c-4 pieces to maximize surface contact with dissociation enzymes in subsequent steps.

For larger samples, the tissue should be divided into batches for mincing and combined prior to addition of dissociation enzymes, collagenase II and DNase I. Dissociation enzymes are incorporated in the protocol to break down the extracellular matrix to yield a single cell suspension. Many types of enzymes are available, such as collagenases, Trypsin, Papain, and HyQTase, among others.

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