Use case

Use A498 in 2D culture to test how compounds or genetic perturbations affect proliferation/survival—baseline solid-tumor readout.

1. 01[Prep] Confirm complete medium per datasheet; mycoplasma-negative before key assays.
2. 02[Seed] Count and plate so cultures are ~40–70% confluent at dosing; overnight for adherent lines.
3. 03[Dose] Vehicle + ≥5-point log dose grid; optional cytotoxic positive control. Keep DMSO ≤0.1–0.5% if possible.
4. 04[Incubate] Typically 24–72 h; minimize disturbance.
5. 05[Viability] MTT / CCK-8 / CellTiter-Glo per kit; ≥3 wells/group.
6. 06[Optional apoptosis] Annexin V / caspase or DNA fragmentation.
7. 07[Optional colony] Low-density plating, 1–3 weeks, crystal violet colony counts.
8. 08[Calculate] % inhibition vs vehicle; fit IC₅₀; ≥2–3 independent repeats.
9. 09[QC] Log passage and serum lot; exclude contaminated/detached wells.

Reagents / compounds

Readouts

(1) Viability / IC₅₀; (2) optional apoptosis; (3) optional colonies; (4) morphology.

Literature & manuals

1. [1]PubMedPMID 327080Journal of the National Cancer Institute · 1977
   One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice.
2. [2]PubMedPMID 375235Proceedings of the National Academy of Sciences of the United States of America · 1979
   Human melanoma cells have both nerve growth factor and nerve growth factor-specific receptors on their cell surfaces.
3. [3]PubMedPMID 6606682J Immunol Methods · 1983
   Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.
4. [4]PubMedPMID 16740937Nat Protoc · 2006
   Clonogenic assay of cells in vitro.
5. [5]PubMedPMID 2135685J Exp Med · 1990
   Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis.

Use case

Assess horizontal migration or matrix invasion of A498 for metastasis-related mechanisms and anti-migratory screens.

Reagents / compounds

Readouts

(1) Wound closure; (2) invaded cell counts; (3) paired viability.

Literature & manuals

1. [1]PubMedPMID 327080Journal of the National Cancer Institute · 1977
   One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice.
2. [2]PubMedPMID 375235Proceedings of the National Academy of Sciences of the United States of America · 1979
   Human melanoma cells have both nerve growth factor and nerve growth factor-specific receptors on their cell surfaces.
3. [3]PubMedPMID 17546025Cell Adh Migr · 2007
   An introduction to the wound healing assay using live-cell microscopy.
4. [4]PubMedPMID 22130855J Vis Exp · 2011
   In vitro cell migration and invasion assays.

Use case

Fix passage window and mycoplasma/morphology baselines to reduce lot drift.

Readouts

Mycoplasma-negative, stable morphology, in-window passage, viability baseline.

Literature & manuals

1. [1]PubMedPMID 327080Journal of the National Cancer Institute · 1977
   One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice.
2. [2]PubMedPMID 375235Proceedings of the National Academy of Sciences of the United States of America · 1979
   Human melanoma cells have both nerve growth factor and nerve growth factor-specific receptors on their cell surfaces.