Use cases
#4 fat-pad HER2+/ER+ BT-474 typically needs exogenous estrogen. This SOP uses Cornelius: 2×10⁷ in 50% Matrigel:50% RPMI; E2 0.72 mg / 90-day 3 days pre—injectate μL not stated. Chen contrast: 4×10⁶ luc-BT-474 into #4 pad, volume/matrix not stated. Not s.c. 1×10⁷ / 100 μL, and not Price 1990 / 231’s 2×10⁶.
Catalog: MC-h028 · Product modeling page · In-vitro spheroid
RUO. IACUC and SPF required. Cell number/volume/anesthetic follow this protocol’s compound card and PMIDs—do not copy MDA-MB-231. IACUC may override published anesthetic numbers. Not a substitute for the veterinary SOP.
Use-case overview
Use this page for local orthotopic growth / anti-HER2 efficacy. Chen s.c. and Price 1990 are not this needle.
| Use | Fit | Primary readout | Notes |
|---|---|---|---|
| #4 fat-pad orthotopic screen | Recommended | Orthotopic volume, pathology[1] | Cornelius: ♀ nude, 2×10⁷ in 50% Matrigel:50% RPMI, #4 pad; E2 0.72 mg / 90-day, 3 d pre; enroll when >95 mm³[1]. μL not stated. |
| luc lower-cell contrast | Suitable | BLI / primary | Chen: 4×10⁶ luc-BT-474 left #4 pad; volume/matrix not stated—not Cornelius’s default[2]. |
| s.c. CDX | Not recommended | — | Use the s.c. protocol. Do not default the fat pad to 100 μL 1:1 gel. |
Does the workflow match?
Fat-pad orthotopic shares this skeleton; s.c. is another path.
| Use | vs foolproof SOP | Differences vs core SOP |
|---|---|---|
| #4 fat-pad orthotopic | Same core SOP | This page’s Cornelius 2×10⁷. |
| s.c. CDX | Other route | Chen 1×10⁷ / 100 μL, not into the pad. |
Host spec: sex × strain × estrogen
Cornelius defaults to female nude + 0.72 mg-class E2 pellet. RUO.
| Indication / context | Sex | Strain / host | Notes |
|---|---|---|---|
| This SOP default: #4 fat-pad orthotopic | Female | Nude (Cornelius / Chen) | 2×10⁷ in 50% Matrigel; E2 0.72 mg / 90-day, 3 d pre[1]. |
In one line
IACUC + estrogen → expand → fat-pad inject → caliper → pathology.
D−1 / D0 timeline
1. D−7 to D−3
Quarantine; confirm sex/strain/endpoints and IACUC.
2. D−1
Feed at 80–90% confluence.
3. D0
Inoculate per the recipe card (Cornelius：2×10⁷ / 50% Matrigel:RPMI（μL 未写）。Chen luc 对照 4×10⁶（体积/基质未写）).
4. After
Follow the monitor log; stop at endpoints.
Protocol overview
Scenario-specific flowchart (core engraftment skeleton with host/therapy or imaging/readout changes).
- IACUC + female nude quarantine
- Implant E2 pellet per IACUC (Cornelius: 3 d pre)
- STR OK; expand parental BT-474
- 2×10⁷ in 50% Matrigel:RPMI (μL from IACUC)
- Slow #4 fat-pad inject
- Caliper until >95 mm³ enroll
- Endpoint primary pathology
Novice pack (literature cases)
Flowchart buttons open the matching table. Full pack below, in use order.
Unpack list (plan per mouse)
SKUs link to catalog items. Follow the MC-h028 datasheet for medium. Matrigel only if the compound card lists it. Anesthetic: compound card/PMID; IACUC may override.[1]
| Item | Per mouse / study | Notes |
|---|---|---|
| BT-474 cells MC-h028 | Start from 1 vial | STR / mycoplasma-qualified; log passage |
| FBS MC100 | Complete medium per datasheet | Prefer regular grade |
| Ca²⁺/Mg²⁺-free PBS / serum-free medium | Resuspend; volume on the recipe card | Per SOP |
| T75 or 10-cm dish | See expansion table | Log-phase harvest; T75 yield planned at ~5×10⁶, not a measured lot value |
| Matrigel (only if this protocol uses it) | Per compound card | On ice; skip if not listed |
| Syringe / needle | 1 per mouse + spares | Gauge on the inject card and the paper |
| Exogenous estrogen (pellet/slow-release, per IACUC) | Implant before inoculum (dorsal flank, away from the inject site) | Chen: 1.7 mg / 90-day, 3 d pre. Lai: 1.7 mg, 7 d pre. Cornelius orthotopic: 0.72 mg / 90-day, 3 d pre. Milligrams on this page are not a default dose. |
Expansion cases: mice vs T75
按本协议出现的最高细胞数 2e+7 规划; dead-space ×1.5. Do not reuse MDA-MB-231 orthotopic 2×10⁶ math.
| Animals | T75 flasks |
|---|---|
| 1 | 6 |
| 2 | 12 |
| 3 | 18 |
| 4 | 24 |
| 5 | 30 |
| 6 | 36 |
| 7 | 42 |
| 8 | 48 |
| 9 | 54 |
| 10 | 60 |
Inoculum recipe
This SOP dose string: Cornelius：2×10⁷ / 50% Matrigel:RPMI（μL 未写）。Chen luc 对照 4×10⁶（体积/基质未写）. Batch ×1.5 for dead space; load one syringe per animal.[1]
| Item | This SOP | Notes |
|---|---|---|
| Cells / volume | Cornelius：2×10⁷ / 50% Matrigel:RPMI（μL 未写）。Chen luc 对照 4×10⁶（体积/基质未写） | Follow the paper’s Methods; pilot n=3–5 |
| Matrix | Only if the compound card lists it | Do not copy another line’s 1:1 or 25% |
| Window | On ice; finish promptly (often 30–60 min) | Resuspend or discard if delayed |
| Viability | ≥90% singles | <90% or clumped: do not inject |
Injection / surgery checklist
#4 mammary fat pad—not flank s.c. Watch this line’s JoVE/paper. Anesthetic: compound card/PMID; IACUC may override.
| Check | Pass | If fail |
|---|---|---|
| Landmark | #4 nipple / fat pad | s.c. bleb: log as failed |
| Push | Slow local bleb; control leak | Immediate leak: log as failed |
| Air / clumps | No air; singles on the scope | Clumped: do not inject |
Culture card (BT-474 / MC-h028)
Inverted microscope (schematic 10× field): confluence is the % of the growth surface covered by cells. If the monolayer is even, that matches the fraction of the field occupied. Split/harvest at the middle panel. Click the schematic to enlarge.
Follow the datasheet. Confluence = % of growth area covered; schematic, not a real micrograph.[1]
| Item | Practice |
|---|---|
| Medium | Datasheet first (MC-h028). Feed before harvest as usual for this line. |
| 80–90% confluence (harvest) | Nearly full with small gaps; no stacking. Too sparse for yield; a packed monolayer should not be injected. |
| Passage | Fix an early–mid window for in-vivo work; validate labeled lines. |
| Day −1 | Feed; check morphology; expand enough T75s. |
What success looks like (expected, not a guarantee)
Kinetics vary by strain, passage, and dose—pilot first. Troubleshoot before raising cell number.[1]
| Time point | Typical appearance |
|---|---|
| Day 0 | Local bleb; no ongoing leak; active after recovery |
| Follow-up window | Orthotopic volume, pathology, optional HER2 markers. |
| Take | Lots vary; pilot n≈3–5 |
Troubleshooting
Welfare issues follow the IACUC SOP—do not improvise doses or anesthetic concentrations from this page.
| Sign | Likely cause | Action |
|---|---|---|
| Clumps / clogged needle | Incomplete dissociation or gel set | Do not inject; re-ice or discard |
| Failed-route signs | Leak or wrong plane | Drop from the primary analysis; use the checklist |
| No readout in the window | Missed inject, dead cells, wrong dose/host | Audit the log and the paper; do not copy another line’s dose |
| Weight drop / distress / ulcer | Burden or surgical complication | Humane endpoint now |
Monitoring log fields
Volume ≈ L × W² / 2. Ulcer and weight outrank “wait a few more days.”
| Field | How to log |
|---|---|
| Date / D0 | Inoculation day |
| Mouse / arm / passage | Lock IDs after randomization |
| Route success | Y/N (reason if no) |
| Body weight (g) | Same day as observations; sharp drop triggers endpoint |
| Caliper | L, W; volume ≈ L×W²/2 |
Literature case comparison
Rows are this protocol’s references only—other lines’ Methods numbers are not copied in. Open the PMID.
- 01[Ethics] IACUC; female immunodeficient mice. RUO. Cornelius/Chen orthotopic do not give inoculum anesthetic mg/%; immobilize per IACUC. Write weight and estrogen-related endpoints.
- 02[Hormone] Implant slow-release estradiol before inoculum (size from the IACUC SOP only). Cornelius: 0.72 mg / 90-day, 3 d pre[1]. Do not treat Chen’s s.c. 1.7 mg as the only correct size.
- 03
- 04[Surgery] Slow #4 mammary fat-pad injection. Not flank s.c. 100 μL.
- 05[Monitor] Cornelius: tumors >95 mm³ eligible for randomization[1].
- 06[Endpoint] Primary pathology. Do not default BT-474 to Price 1990 mets spectra.
Reagents / materials
| Item | Role | Conc. / dose |
|---|---|---|
| BT-474 cells (MC-h028) | Inoculum | Cornelius：2×10⁷ / 50% Matrigel:RPMI（μL 未写）。Chen luc 对照 4×10⁶（体积/基质未写） |
| Matrigel (commonly used) | Engraftment aid | 常 1:1，按 SOP |
| Exogenous estrogen (slow-release pellet / IACUC size) | Support HER2+/ER+ BT-474 engraftment | 规格只来自 IACUC。文献对照：Chen 1.7 mg / 90 天，接种前 3 天；Lai 1.7 mg，接种前 7 天；Cornelius 原位 0.72 mg / 90 天，接种前 3 天。不要把网页毫克数当唯一正确。 |
| Anesthetic / analgesic | Surgery & welfare | Cornelius/Chen 原位未给接种麻醉 mg/%；需要制动时按批件 |
| Test drug / vehicle (optional) | Treatment arm | 按药理方案 |
Readouts
Orthotopic volume, pathology, optional HER2 markers.
References (PubMed)
- [1]PMID 38530115 — Pharmacological Characterization of SDX-7320/Evexomostat: A Novel Methionine Aminopeptidase Type 2 Inhibitor with Anti-tumor and Anti-metastatic Activity. Mol Cancer Ther (2024)
- [2]PMID 30297810 — Ultrasmall targeted nanoparticles with engineered antibody fragments for imaging detection of HER2-overexpressing breast cancer. Nat Commun (2018)
- [3]PMID 212572 — Isolation of two human tumor epithelial cell lines from solid breast carcinomas. J Natl Cancer Inst (1978)
Disclaimer: RUO; IACUC required. Optimize by strain and pilot. Red tags mark weak or non-metastatic parental endpoints.