ISAC Sample Preparation and Handling 2

Question 1

Which fixative is most commonly used to preserve cell surface antigen integrity while allowing subsequent intracellular staining?

Accepted Answer

Paraformaldehyde

Answer

Paraformaldehyde (PFA) at 1-4% preserves surface antigens and cell morphology while still permitting permeabilization for intracellular staining.

Question 2

When preparing peripheral blood mononuclear cells (PBMCs) via density gradient centrifugation, what is the primary purpose of the wash steps after isolation?

Accepted Answer

To remove platelets and residual gradient medium

Answer

Post-isolation washes remove platelets and residual Ficoll/gradient medium, both of which can interfere with antibody staining and instrument performance.

Question 3

A researcher notices high background fluorescence after staining frozen tissue sections for flow cytometry. Which blocking agent is most likely to reduce nonspecific antibody binding?

Accepted Answer

Normal serum from the host species of the secondary antibody

Answer

Normal serum from the same host species as the secondary antibody saturates nonspecific Fc receptor binding sites, reducing background staining.

Question 4

Which enzyme is most frequently used to dissociate solid tumors into single-cell suspensions for flow cytometric analysis?

Accepted Answer

Collagenase

Answer

Collagenase digests collagen fibers in the extracellular matrix, making it the preferred enzyme for dissociating solid tumors while maintaining surface antigen integrity.

Question 5

What is the recommended maximum time between blood collection and staining for surface markers when using whole blood protocols?

Accepted Answer

8 hours

Answer

Whole blood samples should ideally be stained within 8 hours of collection to minimize cellular activation, antigen degradation, and changes in leukocyte subsets.

(ISAC) International Society for the Advancement of Cytometry Practice Test

(ISAC) International Society for the Advancement of Cytometry Practice Test

ISAC Sample Preparation and Handling 2