案例 03：华东师范大学-黄正齐老师样品检测报告（鬼笔环肽·肌动蛋白）
📋 项目概况
客户单位：华东师范大学
项目负责人：黄正齐老师
项目目标：鬼笔环肽（Phalloidin）肌动蛋白（Actin）荧光标记样品的多平台检测评估，覆盖 Acti-Stain 555 Phalloidin 与 Alexa Fluor™ 532 Phalloidin 两款染料，跨 2023 年 4 月至 8 月共 6 轮检测迭代。
Reagents标记原料记录
🎨 Acti-Stain 555 Phalloidin
🎨 Alexa Fluor™ 532 鬼笔环肽
激发/发射波长：531 / 554 nm
产品链接：thermofisher.cn/order/catalog/product/A22282
In DMSO：150 μL 66 μM stock
In methanol：1.5 mL 6.6 μM stock
本项目选择 甲醇溶解，后续需做浓度滴定（titration）以平衡信号强度与背景荧光。
使用说明 PDF：MAN0001777_Phalloidins_UG.pdf ↗
Equipment检测设备：Olympus FV3000
60× 物镜 LSM 激光光源：
20230418Actin-Alexa555 样品检测详情
对比样品：两批 BSC-1 细胞，分别使用 4% PFA 与 4% PFA + 1% GA 两种固定方案；激光仅开 0.3%，评估低激光下两种固定剂的细节保留能力。
| 维度 | 样品 1（S01） | 样品 2（S02） |
|---|---|---|
| 标签 | S01-Alexa555 phalloidin 4% PFA Fix Bright、lowBG — 样品 A |
S02-Alexa555 phalloidin 4% PFA + 1% GA Fix Bright、higher BG、good details — 样品 B |
| 固定剂 | 4% PFA | 4% PFA + 1% GA |
| 标记信息 | Alexa555 phalloidin | Alexa555 phalloidin |
| 样品备注 | 背景更干净，雾状与丝状细节足够，样品非常明亮（0.3% 激光） | 背景较高，但信号更亮，雾状与丝状细节保存更多（0.3% 激光） |
60× 共聚焦成像（S01 vs S02）
20× 低倍概览
Time扫描时间尺度
在 2 μs/pixel 的扫描参数下，整张 1024 × 1024 像素图采集耗时约 3.2 s。该参数兼顾了样品的光漂白控制与微管细节的清晰度，是后续多轮检测迭代的基础设置。
20230601Actin-Alexa532 样品检测详情
标记原料
成像检测结果（FV3000 60×）
| 样品 | S01 | S02 | S03 | S04 |
|---|---|---|---|---|
| 染料 | Alexa Fluor 532 phalloidin | Alexa Fluor 532 phalloidin | Alexa Fluor 532 phalloidin | Alexa Fluor 532 phalloidin |
| 浓度 | 4% | 4% | 50% | 16.6% |
| 孵育 | 4 ℃ 过夜（0602） | 4 ℃ 过夜（0602） | 4 ℃ 过夜（0602） | 4 ℃ 过夜（0602） |
基恩士对照（60×）
20230602Actin-Alexa532 样品检测详情
标记原料
成像检测结果
| 样品 | S01 | S02 | S03 | S04 | S05（+ 标准样） |
|---|---|---|---|---|---|
| 染料 | Alexa Fluor 532 | Alexa Fluor 532 | Alexa Fluor 532 | Alexa Fluor 532 | Alexa Fluor 532 |
| 浓度 | 10% | 50% | 20% | 100% | — |
| 染色时间 | 20230602 | 20230602 | 20230602 | 20230602 | 20230602 |
基恩士初扫（Brightness:1 / 6s）
基恩士复测（Brightness:1 / 12s · 4 ℃ 过夜孵育）
正对照样品（S05）
| Positive control（基恩士） | |
|---|---|
| 曝光时间 | 1 / 10 s |
| 通道 | CH2 |
| Gain | +6 dB |
| 激光强度 | 100% |
20230605Actin-Alexa532 浓度优化 · 关键结论
原料记录
实验条件：环境温度 27 ℃ · 染色时间 3 h
| 样品 | S01 | S02 | S03 ⭐ 最优 | S04 | S05 正对照 |
|---|---|---|---|---|---|
| 染料浓度 | 50% | 100% | 200% | 300% | 488 标准 |
| 配置体系 | 染料（甲醇）12.5 μL + Buffer 12.5 μL |
染料（DMSO）2.5 μL + Buffer 22.5 μL |
染料（DMSO）5 μL + Buffer 20 μL |
染料（DMSO）7.5 μL + Buffer 17.5 μL |
488 1 μL + Buffer 24 μL |
20× 时序 cycle
60× 共聚焦单帧（6.5 日）
60× 532 复测（6.6 日，S03/S04）
从染料自身看，该批次 Alexa 532 染料质量较差，需 50× 于正对照染料浓度才能达到相同效果，建议更换染料。
20230614Actin-Alexa532 样品检测报告
标记原料
成像结果（S1 · 60× 532 + 明场 BF）
20230809Actin-Alexa532 样品检测详情
60× 532 成像
Protocol实验流程
🧪细胞爬片表面处理
- 将洁净的细胞爬片放入六孔板或 3.5 cm 培养皿中，加入 1–2 mL 完全培养基使之浸润爬片；
- 放入培养箱孵育 12 h 以上，使用前吸除培养基。
🧫成像细胞接种
- 取细胞状态最优的培养皿消化处理，消化离心后细胞计数，确保每孔（六孔板）/ 每皿 内有 4 × 10⁴ 个活细胞，计算所需细胞总量；
- 取出所需细胞，按每孔/每皿加 2 mL 培养基计算，配制成细胞悬液并加入到孔板或培养皿中；
- 成像细胞培养需 1–2 天，观测到细胞状态良好且单个细胞舒展不拥挤、面积大、细胞透光性强透明度高时，可进行后续成像步骤。
🎨染色步骤
- 细胞固定：使用 4% PFA 固定细胞形态；
- 背景荧光淬灭：使用硼氢化钠消除背景荧光；
- 细胞封闭：使用 BSA 处理细胞，使其结合位点更易结合染料；
- 细胞染色：使用 Alexa 532 染料染色细胞，过夜孵育；
- 细胞后固定：再次使用 PFA 固定细胞形态，使样片形态更加稳定。
⚠️ 注意：每一步细胞处理后都需要对细胞进行清洗，去除上一步使用的试剂。
Methods染色过程描述
🧬Actin staining（肌动蛋白染色）
Cell culture
The Biologics Standards-Cercopithecus-1 (BSC-1) cell line and NIH/3T3 cell line were purchased from Pricella Life Technology Co. LTD. BSC-1 cells and NIH/3T3 cells were cultured in DMEM (Invitrogen, #11965-118) supplemented with 10% fetal bovine serum (FBS) (Gibco, #16010-159). To prevent bacterial contamination, 100 μg/mL penicillin and streptomycin (Invitrogen, #15140122) were added in the DMEM medium. Cells were grown under standard cell culture conditions (5% CO₂, humidified atmosphere at 37 ℃). BSC-1 cells were plated on #1.5 glass-bottom dish over 48 h before sample preparation. For cell passage, cells were washed with pre-warmed PBS (Life Technologies, #14190500BT) 3 times and digested with 25% trypsin (Gibco, #25200-056) for 30 s. BSC-1 cell line were tested for potential mycoplasma contamination (MycoAlert, Lonza), and all tests showed negative results.
Cell staining
Cells were grown on 35 mm, #1.5 glass coverslips (Standard Imaging (Beijing) Biotech, STGBD-035-1). To increase cell adhesion, we pre-treated glass-bottom dishes with fibronectin (Invitrogen, #33016015) for 1 h at 37 ℃. On the day of sample preparation, the cell density should be about 50%–70%. Cells were fixed with 37 ℃ pre-warmed fixation buffer for 10 min, containing 4% paraformaldehyde (EMS) and 0.1% glutaraldehyde in PBS. Then the sample were washed three times with PBS. For quenching the background fluorescence, we incubate the cells with 2 mL 0.1% NaBH₄ solution in PBS for 7 min, optionally shake on the shaker (<1 Hz). The sample were washed three times with 2 mL PBS and then incubated for 30 min in PBS containing 5% BSA (Jackson, #001-000-162) and 0.5% Triton X-100 (Fisher Scientific) at 37 ℃. All dyes were diluted in the 5% BSA + 0.5% triton solution described above. Next, we incubate the sample for 40 min with the appropriate dilution of dyes: phalloidin-Alexa532 (ThermoFisher, A22282) or Acti-Stain 555 Phalloidin (Cytoskeleton, PHDH1-A) at 25 ℃. After washed 3 times with PBS, cells were fixed with post-fixation buffer for 10 min. The samples were stored in 4 ℃ in PBS no more than one week.
🧬Tubulin staining（微管染色）
Cell culture
The Biologics Standards-Cercopithecus-1 (BSC-1) cell line was purchased from Pricella Life Technology Co. LTD. BSC-1 cells were cultured in DMEM (Invitrogen, #11965-118) supplemented with 10% fetal bovine serum (FBS) (Gibco, #16010-159). To prevent bacterial contamination, 100 μg/mL penicillin and streptomycin (Invitrogen, #15140122) were added in the DMEM medium. Cells were grown under standard cell culture conditions (5% CO₂, humidified atmosphere at 37 ℃). BSC-1 cells were plated on #1.5 glass-bottom dish over 48 h before sample preparation. For cell passage, cells were washed with pre-warmed PBS (Life Technologies, #14190500BT) 3 times and digested with 25% trypsin (Gibco, #25200-056) for 30 s. BSC-1 cell line were tested for potential mycoplasma contamination (MycoAlert, Lonza), and all tests showed negative results.
Immunofluorescence staining
Cells were grown on 35 mm, #1.5 glass coverslips (Standard Imaging (Beijing) Biotech, STGBD-035-1). To increase cell adhesion, we pre-treated glass-bottom dishes with fibronectin (Invitrogen, #33016015) for 1 h at 37 ℃. On the day of sample preparation, the cell density should be about 50%–70%. Cells were fixed with 37 ℃ pre-warmed fixation buffer for 10 min, containing 4% paraformaldehyde (EMS) and 0.1% glutaraldehyde in PBS. Then the sample were washed three times with PBS. For quenching the background fluorescence, we incubate the cells with 2 mL 0.1% NaBH₄ solution in PBS for 7 min, optionally shake on the shaker (<1 Hz). The sample were washed three times with 2 mL PBS and then incubated for 30 min in PBS containing 5% BSA (Jackson, #001-000-162) and 0.5% Triton X-100 (Fisher Scientific) at 37 ℃. All antibodies were diluted in the 5% BSA + 0.5% triton solution described above. Next, we incubate the sample for 12 hours the appropriate dilution of primary antibodies: beta-tubulin (DSHB-E7) at 4 ℃. After primary antibodies incubation, the cells were washed 5 min with 2 mL PBS for three times. Secondary antibodies were incubated for 60 min with the appropriate dilutions of secondary antibodies (Cy3b secondary antibodies were conjugated in our laboratory) at 25 ℃, the sample were protected from light. After washed 3 times with PBS, cells were fixed with post-fixation buffer for 10 min. The samples were stored in 4 ℃ in PBS no more than one week.
Next下一步行动
✅ 已完成：在基恩士上建立样品亮度的 positive control，作为后续批次的对照基准。
| 浓度（甲醇） | 20% | 50% | 75% | 100% | DMSO 溶 | 150% | 200% | 250% | 300% |
|---|---|---|---|---|---|---|---|---|---|
| 对应 μM | 6.6 μM | 66 μM | |||||||
| 是否需要 | 不需要 | 1:1 | 不需要 | 改为 DMSO Stock | — | 不需要 | 不需要 | 不需要 | — |
| phalloidin Alexa532 用量 | 5 μL | 12.5 μL | 20 μL | 25 μL | — | 3.75 μL | 5 μL | 6.25 μL | 7.5 μL |
用量汇总：甲醇配置方案共需 57.5 μL；DMSO 浓缩方案共需 22.5 μL。
建议：将甲醇配置的染料 225 μL 浓缩至 DMSO 配置的 22.5 μL，建议一次性配置 200 μL（→ 实际可用 20 μL）。
—— 案例 03 · 客户案例 · 华东师范大学-黄正齐老师样品检测报告 ——