A. Ion-exchange chromatography and FACS

B. Hydrophobic chromatography and density gradient centrifugation

C. Density gradient centrifugation and FACS

D. Hydrophobic chromatography and FACS

Correct Answer: C. Density gradient centrifugation and FACS

Density gradient centrifugation separates RBCs (denser, pellet) from WBCs (lighter, interface layer), while FACS sorts both populations by light scatter (size/complexity) and specific markers.

Option Analysis

A. Ion-exchange chromatography and FACS
Ion-exchange separates charged molecules/proteins by charge, not intact cells. Cells have complex surface charges making chromatography impractical for blood cell separation.

B. Hydrophobic chromatography and density gradient centrifugation
Hydrophobic chromatography purifies proteins/lipids by hydrophobicity. Intact blood cells don’t separate by surface hydrophobicity; both RBC/WBC membranes are amphipathic.

C. Density gradient centrifugation and FACS (Correct)

• Density gradient: RBCs (1.09-1.10 g/mL) pellet through Ficoll (1.077 g/mL); WBCs collect at interface. Standard PBMC isolation method.

• FACS: Forward scatter separates RBC (small) vs WBC (large); side scatter distinguishes granulocytes (high granularity) from lymphocytes (low).

D. Hydrophobic chromatography and FACS
Hydrophobic chromatography unsuitable for intact cells (same issue as B).

Density gradient centrifugation and FACS provide gold-standard red vs white blood cell separation—essential techniques for immunology and GATE Life Sciences hematology.

Standard Protocol

Step 1: Ficoll-Hypaque (1.077 g/mL) centrifugation yields RBC pellet, WBC interface layer (15 min, 400g).
Step 2: FACS sorts lymphocytes (CD45+SSC low), granulocytes (CD45+SSC high), RBCs (CD71+Ter119+ FSC low).

Method Comparison

GATE Applications

Tests cell separation principles in immunology, hematology. Ficoll isolation + flow cytometry = standard workflow for functional cell studies.