Use cases
Parental orthotopic mammary fat-pad dose is not locked to a PubMed-checkable default SOP. Pilot required. Do not copy s.c. 5×10⁷.
Catalog: MC-h029 · Product modeling page
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
Honestly marks not locked; s.c. is another protocol.
| Use | Fit | Primary readout | Notes |
|---|---|---|---|
| Parental orthotopic | Not recommended | — | Dose not locked. |
| 5×10⁷ s.c. CDX | Recommended | See s.c. protocol | Use the s.c. page. |
Does the workflow match?
Unlocked orthotopic and s.c. are not interchangeable.
| Use | vs foolproof SOP | Differences vs core SOP |
|---|---|---|
| Orthotopic (unlocked) | Other route | Pilot required. |
| s.c. CDX | Same core SOP | 5×10⁷ ♀ BALB/c nude[1]. |
Host spec
Orthotopic parental not locked.
| Indication / context | Sex | Strain / host | Notes |
|---|---|---|---|
| Orthotopic | ♀ / per IACUC | Not locked | Pilot. |
In one line
Pilot → lock SOP.
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 (原位：亲本剂量未锁).
4. After
Follow the monitor log; stop at endpoints.
Protocol overview
Scenario-specific flowchart (core engraftment skeleton with host/therapy or imaging/readout changes).
- Pilot
- Fix passage
- —
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-h029 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-549 cells MC-h029 | 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 |
Expansion cases: mice vs T75
按本协议出现的最高细胞数 5e+7 规划; dead-space ×1.2. Do not reuse MDA-MB-231 orthotopic 2×10⁶ math.
| Animals | T75 flasks |
|---|---|
| 1 | 12 |
| 2 | 24 |
| 3 | 36 |
| 4 | 48 |
| 5 | 60 |
| 6 | 72 |
| 7 | 84 |
| 8 | 96 |
| 9 | 108 |
| 10 | 120 |
Inoculum recipe
This SOP dose string: 原位：亲本剂量未锁. Batch ×1.2 for dead space; load one syringe per animal.[1]
| Item | This SOP | Notes |
|---|---|---|
| Cells / volume | 原位：亲本剂量未锁 | 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
Follow published parameters for this line—do not copy other lines. Anesthetic: compound card/PMID; IACUC may override.
| Check | Pass | If fail |
|---|---|---|
| Route | Match the name/IACUC SOP | Unsure: read the PMID; do not guess |
| Air / clumps | No air; singles on the scope | Clumped: do not inject |
Culture card (BT-549 / MC-h029)
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-h029). Feed before harvest as usual for this line. |
| Doubling time | ~3.7 days (Cellosaurus; not a measured lot) |
| 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 | Route checks passed; active after recovery |
| Follow-up window | Per pilot design. |
| 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
Often no caliper volumes on this route. Weight and IACUC endpoints outrank the image.
| 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 |
| Readout | Per pilot design. |
Literature case comparison
Rows are this protocol’s references only—other lines’ Methods numbers are not copied in. Open the PMID.
| Paper | Year | Journal | How to use |
|---|---|---|---|
| — | Parental orthotopic fat pad | Dose not locked | Pilot required |
- 01[Note] Parental orthotopic dose not locked.
- 02[Contrast] s.c. 5×10⁷ is on the other protocol[1].
Reagents / materials
| Item | Role | Conc. / dose |
|---|---|---|
| BT-549 cells (MC-h029) | Inoculum | 原位：亲本剂量未锁 |
| Anesthetic / analgesic | Surgery & welfare | 按批件 |
| Test drug / vehicle (optional) | Treatment arm | 按药理方案 |
Readouts
Per pilot design.
References (PubMed)
- [1]PMID 41307753 — Lon peptidase 1 promotes proliferation and metastasis in breast cancer via interleukin 6 signaling. Discov Oncol (2025)
Disclaimer: RUO; IACUC required. Optimize by strain and pilot. Red tags mark weak or non-metastatic parental endpoints.