# CRO METHODS SHEET (1 PAGE)
**Study**: Raft disruption vs direct P-gp block — paclitaxel accumulation and sensitization in TNBC PacR  
**Hypothesis (Test, Do Not Assume)**: $C_{\text{raft}}\downarrow \implies J_{\text{P-gp}}\downarrow \implies F(t=1\,\text{h})\uparrow \implies \text{IC}_{50}^{\text{231-PacR}}\downarrow$, with MCF-10A viability $\ge 80\%$ at the PacR $\text{IC}_{50}$ exposure.  
**Not in Scope**: In vivo, clinical claim, bound-MT occupancy (no assay on this sheet). $B_{\text{MT}}$ remains a model term only.

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### 1. Cells
| Line | Role | Source | Notes |
|---|---|---|---|
| **MDA-MB-231-PacR** | Acquired PTX-resistant TNBC | CRO stock or Altogen / Creative Biolabs | Document selection history; confirm ABCB1/P-gp by WB before dosing |
| **MDA-MB-231-P** | Parental sensitive | ATCC HTB-26 | Same medium family as PacR if possible |
| **MCF-10A** | Non-tumorigenic mammary gate | ATCC CRL-10317 | MEGM + specified supplements; no PTX in maintenance |

*QC*: Mycoplasma-negative; STR-authenticated; PacR kept off drug $\ge 5–7\text{ d}$ before assays. Seed so all three lines are 70–80% confluent at treatment.

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### 2. Treatments
* **Vehicle**: 0.1% DMSO.
* **PTX (Viability)**: 0.1, 0.3, 1.0, 3.3, 10, 33, 100, 1000 nM; 72 h; $n=3$ wells $\times \ge 2$ independent plates.
* **PTX (1 h Accumulation)**: 100 nM, $t^* = 1.0\text{ h}$.
* **MβCD (Raft / Cholesterol Pull)**: 1.2 and 2.0 mM, 30 min pre-incubation in serum-free medium, wash, then PTX. Include MβCD-alone arms (no PTX) for LDH + viability.
* **Verapamil (+ Control, Direct ABCB1 Block)**: 10 µM, 30 min pre then co-present with PTX (standard complete P-gp in-vitro block).
* **Arms (All 3 lines)**: Vehicle | MβCD 1.2 | MβCD 2.0 | Verapamil 10 µM | MβCD 2.0 + Verapamil (interaction).
* **Stop Rule**: If MβCD-alone drops any line $<80\%$ viable or LDH $>2\times$ vehicle at 30 min + wash + 72 h, report as lytic and do not interpret that dose as "raft-specific."

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### 3. Assays & 3 Required Plots
* **Plot 1 — $J_{\text{P-gp}}$ (Surrogate Efflux)**:
  * Rho-123 or Calcein-AM accumulation, flow cytometry, 30–60 min substrate after same pre-treatments (no PTX required).
  * Report: median fluorescence, fold vs vehicle, $\pm$ verapamil. Label surrogate, not paclitaxel flux.
* **Plot 2 — $F(t^*)$ / Retention (LC-MS/MS)**:
  * 100 nM PTX, 1.0 h, ice-cold wash $\times 3$, count cells, lyse, spike docetaxel or PTX-d5, extract, LC-MS/MS.
  * Report: pmol / $10^6$ cells and nM in lysate. Operational name: extractable intracellular PTX at 1 h. Include medium leftover PTX.
* **Plot 3 — 72 h $\text{IC}_{50}$ (Viability & Host Gate)**:
  * CellTiter-Glo or MTS; same reader for all lines. Fit 4-parameter log-logistic.
  * Overlay: 231-PacR, 231-P, MCF-10A, each arm. Mark MCF-10A 80% viability on PacR $\text{IC}_{50}$ concentration (gate).

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### 4. Stats & Decision Rules
* $n=3$ technical, $\ge 2$ biological replicates. Mean $\pm$ SD. $\text{IC}_{50}$ 95% CI. Compare PacR arms by extra-sum-of-squares or ANOVA on $\log\text{-IC}_{50}$ and $\log\text{-PTX}$ accumulation.
* **Support Hypothesis If (in PacR only)**: Dye retention $\uparrow$ and 1 h PTX $\uparrow$ and $\text{IC}_{50} \downarrow$, with MCF-10A $\ge 80\%$ at that exposure.
* **Fail If**: MβCD cuts $\text{IC}_{50}$ without changing $J$ or 1 h PTX (off-target/lysis); parental 231 shifts as much as PacR (not P-gp-specific); verapamil does nothing in PacR (wrong model).

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### 5. Deliverables
Raw FCS, LC-MS peak tables, plate maps, viability matrices, fitted $\text{IC}_{50}$ table, the three plots above, STR/mycoplasma certificates, lot numbers.