人恶性黑色素瘤细胞 disease models

A375 (Amelanotic melanoma). Standardized family protocols with foolproof steps, flowcharts, and methodology PubMed refs—optimize per datasheet and pilots. Research use only (RUO).

Adapted from PubMed literature for research use only (RUO); adjust per lab SOP and pilots.

Use case

Use A375 in 2D culture to test how compounds or genetic perturbations affect proliferation/survival—baseline solid-tumor readout.

In one line

Stable culture → seed overnight → dose matrix → timed viability/apoptosis → compare vs vehicle.

Protocol overview

01
Expand; log passage
02
Seed plates to assay density
03
Attach / stabilize overnight
04
Add dose matrix + vehicle
05
Incubate to endpoint
06
Viability / apoptosis assay
07
Compute IC₅₀ or % inhibition

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

Reagents / compounds

Name	Role	Conc.
Test compound / inhibitor	Drug response or pathway intervention	对数梯度；按文献与预实验优化
Vehicle control (e.g. DMSO)	Rule out vehicle artifacts	与最高药组等体积分数
Viability reagent (MTT / CCK-8 / CTG)	Quantify viability/metabolic activity	按试剂盒说明书

Readouts

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

Literature & manuals

[1]PubMedPMID 77569Tissue antigens · 1978
Tissue typing of cells in culture. III. HLA antigens of established human cell lines. Attempts at typing by the mixed hemadsorption technique.
[2]PubMedPMID 265522Proceedings of the National Academy of Sciences of the United States of America · 1977
Nerve growth factor receptors on human melanoma cells in culture.
[3]PubMedPMID 6606682J Immunol Methods · 1983
Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.
[4]PubMedPMID 16740937Nat Protoc · 2006
Clonogenic assay of cells in vitro.
[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 A375 for metastasis-related mechanisms and anti-migratory screens.

In one line

Confluent scratch or Transwell seeding → treat → timed imaging / count invaded cells.

Protocol overview

01
Seed to confluence or Transwell
02
Scratch or add invasion matrix
03
Switch to drug + low-serum medium
04
Timed imaging / stain membrane
05
Quantify wound area or invaded cells
06
Compare vs vehicle

Reagents / compounds

Name	Role	Conc.
Low-serum medium	Reduce proliferation confounders	常用 0.5–2% FBS（按 SOP）
Matrigel / invasion matrix (optional)	Model ECM invasion	按说明书稀释铺胶
Test compound / inhibitor	Drug response or pathway intervention	对数梯度；按文献与预实验优化
Vehicle control (e.g. DMSO)	Rule out vehicle artifacts	与最高药组等体积分数

Readouts

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

Literature & manuals

[1]PubMedPMID 77569Tissue antigens · 1978
Tissue typing of cells in culture. III. HLA antigens of established human cell lines. Attempts at typing by the mixed hemadsorption technique.
[2]PubMedPMID 265522Proceedings of the National Academy of Sciences of the United States of America · 1977
Nerve growth factor receptors on human melanoma cells in culture.
[3]PubMedPMID 17546025Cell Adh Migr · 2007
An introduction to the wound healing assay using live-cell microscopy.
[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.

In one line

Log thaw passage → mycoplasma check → archive morphology → run key assays in-window.

Protocol overview

01
Thaw & log passage
02
Mycoplasma check
03
Archive morphology
04
Run key assays in window

Readouts

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

Literature & manuals

[1]PubMedPMID 77569Tissue antigens · 1978
Tissue typing of cells in culture. III. HLA antigens of established human cell lines. Attempts at typing by the mixed hemadsorption technique.
[2]PubMedPMID 265522Proceedings of the National Academy of Sciences of the United States of America · 1977
Nerve growth factor receptors on human melanoma cells in culture.

Amelanotic melanoma-related 2D viability / drug screen — foolproof protocol

Use case

In one line

Foolproof steps (9)

Reagents / compounds

Readouts

Literature & manuals

Migration / invasion — foolproof protocol (scratch or Transwell)

Use case

In one line

Foolproof steps (8)

Reagents / compounds

Readouts

Literature & manuals

Passage-window & QC checklist

Use case

In one line

Foolproof steps (6)

Readouts

Literature & manuals