1. During the first week post-thawing and subsequent culturing, SK-BR-3 cells may exhibit loosely adherent or floating phenotypes.

2. SK-BR-3 cells recover slowly from cryopreservation and may remain non-adherent initially, which is normal.

3. Post-thawing, the cells require time to regain normal growth; floating cells are common and expected.

4. Loose attachment and floating cells are typical in the first week and after each subculture.

5. If excessive floating cells are observed (especially in the first week), avoid disturbing the culture for several days post-thawing.

6. Upon receipt, assess cell density for subculture. If subcultured, refrain from handling the cells for 3 days (unless medium discoloration occurs). Proper processing improves cell spreading.

7. For persistent floating cells, check viability with trypan blue. Centrifuge gently (125 ×g, 5–7 min) to retain viable floating cells and reintroduce them to the original container during medium changes—do not discard.

8. Initially, cells attach as small clusters or patches, with many remaining suspended. Over days, adherent cells will extend outward. Cell debris may appear and can be removed via medium replacement.

Critical Notes for SK-BR-3 Culture

1. Floating cells: Always recover via gentle centrifugation (125 ×g, 5–7 min) and return them to the culture after medium changes or subculturing.

2. Clustering tendency: Avoid trypsinization until cells reach 70–80% confluency. Overgrowth leads to detachment and floating.

3. Viable floating cells: Collect and reseed them; never discard.

4. Subculture frequency: Passage once weekly. If density is low, a 1:1 split after one week improves morphology.