Cell Biology · Advanced: Introduction & Imaging
08 — Cell Isolation and Flow Cytometry
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Why this matters
Biochemical analysis of organelles and proteins requires isolating specific cell populations or subcellular fractions. Flow cytometry enables high-throughput single-cell analysis; FACS physically separates cells based on fluorescence.
The college version
Core Explanation
Differential Centrifugation
Cells are homogenized (broken open) under gentle conditions that preserve organelle integrity. The homogenate is subjected to sequential centrifugation at increasing speeds:
- Low speed (~600 × g, 10 min): Pellet = nuclei, unbroken cells, large debris
- Medium speed (~10,000 × g, 10–20 min): Pellet = mitochondria, lysosomes, peroxisomes
- High speed (~100,000 × g, 1–2 hours): Pellet = microsomes (ER fragments, plasma membrane vesicles, small vesicles)
- Supernatant: Cytosol (soluble proteins)
Critical caveat: Differential centrifugation produces enriched fractions, not pure organelles. Each pellet is contaminated with other organelles of similar sedimentation properties. Further purification (density gradient centrifugation, immunoisolation) is required for pure preparations.
Flow Cytometry
Flow cytometry analyzes single cells in a fluid stream as they pass through a laser beam, one at a time, at rates of thousands of cells per second.
Measured parameters:
- Forward scatter (FSC): Correlates roughly with cell size — larger cells scatter more light forward
- Side scatter (SSC): Correlates with granularity/internal complexity — granular cells (granulocytes) scatter more light at 90°
- Fluorescence: Cells labeled with fluorophore-conjugated antibodies or expressing fluorescent proteins emit at characteristic wavelengths; multiple fluorophores can be detected simultaneously using different detectors
Gating: The user defines regions (gates) on FSC/SSC or fluorescence plots to select subpopulations of interest for further analysis.
Data output: Histograms (one parameter) or dot plots (two parameters) showing the distribution of cells.
FACS (Fluorescence-Activated Cell Sorting)
FACS is a specialized implementation of flow cytometry that physically separates cells. After analysis, the fluid stream is vibrated to form droplets, each containing (ideally) a single cell. Droplets meeting user-defined criteria are electrically charged and deflected into collection tubes.
FACS can sort based on: Cell size, granularity, fluorescence intensity (single or multiple colors), or combinations of parameters.
Key distinction: All FACS is flow cytometry, but not all flow cytometry is FACS. Many instruments analyze only; FACS instruments analyze AND sort.
Eli explains
The same idea, in plain words
Explain it like I’m 10
If you want to study mitochondria, you cannot just pull them out of a cell one by one with tweezers. Instead, scientists blend up cells and spin the soup in a centrifuge — like a super-fast salad spinner. Heavier things (nuclei) sink first at low speed. Lighter things (mitochondria) sink next at higher speed. The tiniest pieces (membrane fragments) need the highest speed. But it is never perfectly clean — each pile always has a little bit of other stuff mixed in.
Flow cytometry is like a high-speed checkout scanner for cells: thousands of cells fly past a laser one at a time, and the machine measures how big they are, how grainy, and what color they glow. If you want to collect only the green-glowing cells, FACS can do that — it gives each green cell an electric charge and flicks it into a separate tube.
Key takeaways
- High Yield: Differential centrifugation enriches organelles — it does not purify them. Contaminating organelles are always present.
- High Yield: FSC ≈ size; SSC ≈ granularity. Use these to distinguish lymphocytes (small, low SSC) from granulocytes (larger, high SSC).
- High Yield: FACS = flow cytometry + physical cell sorting. The "S" stands for sorting.
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- After differential centrifugation, a researcher calls her 10,000 × g pellet "pure mitochondria." Why is this misleading?
- A blood sample is analyzed by flow cytometry. How would you expect lymphocytes and neutrophils to differ in FSC and SSC?
- What distinguishes a FACS instrument from a standard flow cytometer?
- The 10,000 × g pellet is enriched for mitochondria but also contains lysosomes, peroxisomes, and fragments of other organelles with similar sedimentation rates. True purification requires additional steps (density gradients, immunoisolation).
- Lymphocytes: small (low FSC), not granular (low SSC). Neutrophils: larger (medium FSC), highly granular (high SSC — many cytoplasmic granules).
- FACS instruments can physically sort cells into separate collection tubes based on measured parameters. Standard flow cytometers analyze cells but do not sort them.
Study tools & related lessonsYou’ll learn to · Related
You’ll learn to
- Explain differential centrifugation for organelle enrichment
- Distinguish enrichment from purification
- Describe the principles of flow cytometry (FSC, SSC, fluorescence)
- Explain FACS and its sorting capability
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