Home › Cell Biology › Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment
Cell Biology JoVE (Open Access) Citable · DOI

Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment

DOI: 10.3791/60271-v
What you'll learn
  • ✓ Isolate and enrich pachytene spermatocytes and spermatids from mouse testis
  • ✓ Apply discontinuous BSA density gradient centrifugation using standard equipment
  • ✓ Collect and analyze enriched germ cell fractions by morphology
Protocol

Biopharma Insights Presented here is a protocol for enriching pachytene spermatocytes, round spermatids, and elongating spermatids from adult mouse testes using a discontinuous bovine serum albumin density gradient with standard laboratory equipment.

Difficulty
intermediate
Total time
~2–3 hours per mouse (including dissection, gradient setup, centrifugation, and collection)
Model organism
Mouse (adult male)
Biosafety
BSL-1

Steps

1
Dissect mouse testis and prepare germ cell suspension

Remove testes from adult male mice, mechanically dissociate tissue, and generate a single-cell suspension of germ cells. This prepares the starting material for density gradient separation.

▶ 00:58
2
Load germ cells onto discontinuous BSA density gradient

Apply the prepared germ cell suspension to a discontinuous bovine serum albumin gradient column. Centrifuge to separate cells by density.

▶ 04:50
3
Collect enriched germ cell fractions from gradient

Harvest distinct cell fractions from the gradient corresponding to pachytene spermatocytes, round spermatids, and elongating spermatids based on sedimentation distance.

▶ 06:02
4
Analyze cell fractions by morphology and enrichment

Examine collected fractions using microscopy to confirm cell type identity, viability, and enrichment purity. Document cell morphology and developmental stage markers.

▶ 06:52
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