适用场景

用 A375 在二维培养下评估化合物或基因扰动对增殖/存活的影响，是实体瘤体外机制与药敏的基础读出。

1. 01【准备】确认完全培养液与生长条件（与随货说明书核对）；关键实验前支原体抽检合格。
2. 02【铺板】消化计数，按预实验密度接种（常见使加药时 40–70% 汇合）；贴壁株过夜，悬浮株按密度接种后即可处理。
3. 03【加药】设置溶剂对照与 ≥5 点对数浓度梯度；阳性对照药可选。DMSO 终浓度建议 ≤0.1–0.5%（按 SOP）。
4. 04【孵育】常用 24–72 h（依化合物与课题）；期间避免频繁开门晃板。
5. 05【活力】用 MTT / CCK-8 / CellTiter-Glo 等按说明书检测；每组 ≥3 复孔。
6. 06【可选凋亡】Annexin V / Caspase 或 DNA 片段化，区分抑制增殖与诱导死亡。
7. 07【可选克隆】低密度接种后长期培养（约 1–3 周）结晶紫染色计克隆。
8. 08【计算】抑制率 = 1 − (处理/对照)；拟合 IC₅₀；独立重复 ≥2–3 次。
9. 09【质控】记录代次与血清批次；异常孔剔除并备注。

试剂 / 化合物

读出指标

① 活力曲线 / IC₅₀；② 可选凋亡；③ 可选克隆形成；④ 形态。

文献与说明书

1. [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. [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. [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.

适用场景

评估 A375 的水平迁移或基质侵袭能力，常用于转移相关机制与抗迁移药物筛选。

试剂 / 化合物

读出指标

① 划痕愈合率；② 穿膜细胞数；③ 平行活力。

文献与说明书

1. [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. [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. [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.

适用场景

固定代次与支原体/形态基线，减少批次间漂移。

读出指标

支原体阴性、形态稳定、代次在窗口内、活力基线。

文献与说明书

1. [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. [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.