ISAC Sample Preparation and Handling 3

Question 1

A viability dye that is excluded from live cells but penetrates dead cells with compromised membranes is classified as:

Accepted Answer

A membrane-impermeant viability dye

Answer

Membrane-impermeant dyes like propidium iodide or 7-AAD rely on exclusion from intact membranes, staining only dead or dying cells with compromised membranes.

Question 2

Which centrifugation speed and time is most appropriate for pelleting lymphocytes without damaging cell membranes?

Accepted Answer

400 × g for 10 minutes

Answer

300–400 × g for 8–10 minutes is the standard range for gentle lymphocyte pelleting that avoids mechanical damage while achieving adequate recovery.

Question 3

When collecting bone marrow for flow cytometric immunophenotyping, why is it important to use EDTA rather than heparin as an anticoagulant?

Accepted Answer

Heparin can cause non-specific binding that interferes with CD34 staining

Answer

Heparin can bind to CD34 and other hematopoietic progenitor antigens, causing non-specific staining artifacts that complicate immunophenotyping of stem cell populations.

Question 4

The 'hook effect' in flow cytometry panels occurs when:

Accepted Answer

Excess antibody saturates both specific and non-specific binding sites

Answer

The hook effect occurs when antibody is present in excess, leading to reduced mean fluorescence intensity (MFI) as excess antibody competes with bound antibody.

Question 5

For optimal staining of cytokines using intracellular flow cytometry, cells must be treated with a protein transport inhibitor such as brefeldin A or monensin. What is the purpose of this step?

Accepted Answer

To block cytokine secretion so cytokines accumulate intracellularly

Answer

Brefeldin A and monensin block the Golgi apparatus secretory pathway, causing cytokines to accumulate intracellularly where they can be detected by antibody staining.

(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 3