Use cases
Human TNBC BT-549 s.c. CDX. This SOP uses the LONP1 paper’s control arm: 5×10⁷/mouse, ♀ BALB/c nude s.c.; μL/gel not stated. Not orthotopic fat pad. Do not copy MDA-MB-231’s 2×10⁶ / 100 μL.
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
This page is s.c. CDX only (control-arm dose). Orthotopic fat-pad parental dose not locked.
Does the workflow match?
5×10⁷ is higher than many TNBC recipes; do not swap with 231.
| Use | vs foolproof SOP | Differences vs core SOP |
|---|---|---|
| s.c. CDX | Same core SOP | This page’s 5×10⁷ ♀ BALB/c nude (control arm). |
| Orthotopic | Other route | Parental dose not locked. |
Host spec: sex × strain
Paper used ♀ BALB/c nude. RUO.
| Indication / context | Sex | Strain / host | Notes |
|---|---|---|---|
| This SOP default: s.c. CDX | Female | BALB/c nude | 5×10⁷[1]; μL not stated. |
In one line
IACUC → expand → s.c. 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 (Discov Oncol 对照臂：5×10⁷ ♀ BALB/c nude 皮下（μL/胶未写）).
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
- Expand BT-549 (control framing)
- 5×10⁷
- s.c. bleb
- Caliper
- Endpoint 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-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: Discov Oncol 对照臂：5×10⁷ ♀ BALB/c nude 皮下（μL/胶未写）. Batch ×1.2 for dead space; load one syringe per animal.[1]
| Item | This SOP | Notes |
|---|---|---|
| Cells / volume | Discov Oncol 对照臂：5×10⁷ ♀ BALB/c nude 皮下（μL/胶未写） | 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
Flank s.c. bleb; avoid muscle. Anesthetic: compound card/PMID; IACUC may override.
| Check | Pass | If fail |
|---|---|---|
| Site | Flank s.c. bleb | Intramuscular: log as failed |
| Needle | Often 25–27G; follow the paper | Do not assume orthotopic tuberculin dead space |
| Matrix | Only if this protocol’s compound card lists Matrigel | Never put Matrigel on an i.v. SOP |
| 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 | Local bleb; no ongoing leak; active after recovery |
| Follow-up window | s.c. tumor volume, pathology. |
| 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.
| Paper | Year | Journal | How to use |
|---|---|---|---|
| Discov Oncol 2025[1] | ♀ BALB/c nude | 5×10⁷ (μL not stated) | This SOP default (control) |
- 01[Ethics] IACUC; immunodeficient mice. RUO. Tumor-burden ceiling per IACUC (paper max diameter 15 mm / ~1500 mm³)[1].
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 - 02[Cells] BT-549 (MC-h029) control-arm framing. STR/mycoplasma-qualified. TNBC; do not mix with 231.
- 03[Suspension] This SOP 5×10⁷/mouse[1]. μL/gel not stated. Do not copy 231’s 2×10⁶ / 100 μL.
This SOP dose string: Discov Oncol 对照臂：5×10⁷ ♀ BALB/c nude 皮下（μL/胶未写）. Batch ×1.2 for dead space; load one syringe per animal.[1]
Item This SOP Notes Cells / volume Discov Oncol 对照臂：5×10⁷ ♀ BALB/c nude 皮下（μL/胶未写） 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 Flank s.c. bleb; avoid muscle. Anesthetic: compound card/PMID; IACUC may override.
Check Pass If fail Site Flank s.c. bleb Intramuscular: log as failed Needle Often 25–27G; follow the paper Do not assume orthotopic tuberculin dead space Matrix Only if this protocol’s compound card lists Matrigel Never put Matrigel on an i.v. SOP Air / clumps No air; singles on the scope Clumped: do not inject - 04[Inject] s.c. (exact flank not stated)[1].
- 05[Monitor] Caliper ~every 3 days[1].
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 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 - 06[Endpoint] Tumor pathology.
Reagents / materials
| Item | Role | Conc. / dose |
|---|---|---|
| BT-549 cells (MC-h029) | Inoculum | Discov Oncol 对照臂：5×10⁷ ♀ BALB/c nude 皮下（μL/胶未写） |
| Anesthetic / analgesic | Surgery & welfare | 皮下未给接种麻醉 mg/%；需要制动时按批件 |
| Test drug / vehicle (optional) | Treatment arm | 按药理方案 |
Readouts
s.c. tumor volume, pathology.
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.