Use cases
HER2+/ER+ BT-474 flank s.c. CDX usually needs exogenous estrogen and Matrigel for anti-HER2 pharmacology. This SOP uses Chen: 1×10⁷ in 100 μL PBS:Matrigel 1:1; E2 1.7 mg / 90-day pellet 3 days pre. Lai contrast: same cell number in 200 μL GFR Matrigel, E2 1.7 mg 7 days pre. Not fat-pad orthotopic. Do not copy MDA-MB-231’s 2×10⁶ / 100 μL, and do not treat Price 1990 as a BT-474 dose.
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
This page is local s.c. CDX only. #4 fat-pad orthotopic is another protocol. Price 1990 is MDA-MB-231/435—not BT-474.
| Use | Fit | Primary readout | Notes |
|---|---|---|---|
| Anti-HER2 s.c. screen / caliper | Recommended | s.c. volume, weight, pathology[1] | Chen: ♀ nude 6–8 wk, 1×10⁷ in 100 μL PBS:Matrigel 1:1, right flank; E2 1.7 mg / 90-day, 3 d pre[1]. |
| 200 μL GFR Matrigel contrast | Suitable | Same caliper; nodules by day 14–21 | Lai: 1×10⁷ in 200 μL GFR Matrigel; E2 1.7 mg 7 d pre; volume ≈ L×W²/2[2]. Not Chen’s 100 μL. |
| Mammary fat-pad orthotopic | Not recommended | — | Use the orthotopic protocol. Do not copy Chen’s s.c. 1×10⁷ / 100 μL into the fat pad. |
Does the workflow match?
Anti-HER2 screens share this s.c. skeleton; the fat pad is a different protocol.
| Use | vs foolproof SOP | Differences vs core SOP |
|---|---|---|
| Anti-HER2 s.c. screen | Same core SOP | This page’s Chen 1×10⁷ / 100 μL. |
| Fat-pad orthotopic | Other route | Cornelius 2×10⁷ or Chen luc 4×10⁶, not s.c. 100 μL. |
Host spec: sex × strain × estrogen
Human BT-474 uses immunodeficient females + exogenous estrogen. RUO.
| Indication / context | Sex | Strain / host | Notes |
|---|---|---|---|
| This SOP default: s.c. anti-HER2 screen | Female | Nude (Chen / Lai) | Chen: 1×10⁷ in 100 μL 1:1 Matrigel; E2 1.7 mg / 90-day, 3 d pre[1]. |
| Male | Not recommended | — | Mammary HER2 models are female-standard. |
In one line
IACUC + estrogen plan → expand → s.c. (Matrigel) → caliper → optional therapy → 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 (Chen：1×10⁷ / 100 μL PBS:Matrigel 1:1。Lai 对照 1×10⁷ / 200 μL GFR Matrigel).
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
- STR/mycoplasma OK; expand parental BT-474
- Implant E2 pellet per IACUC (Chen: 3 d pre)
- Count (≥90% viable)
- 1×10⁷ in 100 μL PBS:Matrigel 1:1 (Chen)
- Flank s.c. bleb
- Weekly caliper + weight
- Randomize at set volume
- Endpoint tumor 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 1e+7 规划; dead-space ×1.2. Do not reuse MDA-MB-231 orthotopic 2×10⁶ math.
| Animals | T75 flasks |
|---|---|
| 1 | 3 |
| 2 | 5 |
| 3 | 8 |
| 4 | 10 |
| 5 | 12 |
| 6 | 15 |
| 7 | 17 |
| 8 | 20 |
| 9 | 22 |
| 10 | 24 |
Inoculum recipe
This SOP dose string: Chen：1×10⁷ / 100 μL PBS:Matrigel 1:1。Lai 对照 1×10⁷ / 200 μL GFR Matrigel. Batch ×1.2 for dead space; load one syringe per animal.[1]
| Item | This SOP | Notes |
|---|---|---|
| Cells / volume | Chen：1×10⁷ / 100 μL PBS:Matrigel 1:1。Lai 对照 1×10⁷ / 200 μL GFR Matrigel | 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-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 | s.c. tumor volume/weight, body weight, 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.
- 01[Ethics] IACUC; female immunodeficient mice. RUO. Chen/Lai s.c. do not give anesthetic mg/%; immobilize per IACUC. Write ulcer, weight, and estrogen-related humane endpoints.
- 02
- 03
- 04
- 05[Inject] Flank s.c. bleb; avoid muscle. Keep the pellet away from the inject site.
This SOP dose string: Chen：1×10⁷ / 100 μL PBS:Matrigel 1:1。Lai 对照 1×10⁷ / 200 μL GFR Matrigel. Batch ×1.2 for dead space; load one syringe per animal.[1]
Item This SOP Notes Cells / volume Chen：1×10⁷ / 100 μL PBS:Matrigel 1:1。Lai 对照 1×10⁷ / 200 μL GFR Matrigel 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 - 06[Monitor] Volume ≈ L×W²/2 (Lai; this site’s log)[2].
- 07[Endpoint] Tumor pathology. Mets are not an s.c. success criterion.
Reagents / materials
| Item | Role | Conc. / dose |
|---|---|---|
| BT-474 cells (MC-h028) | Inoculum | Chen：1×10⁷ / 100 μL PBS:Matrigel 1:1。Lai 对照 1×10⁷ / 200 μL GFR Matrigel |
| 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 | Chen/Lai 皮下未给麻醉 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/weight, body weight, pathology.
References (PubMed)
- [1]PMID 30297810 — Ultrasmall targeted nanoparticles with engineered antibody fragments for imaging detection of HER2-overexpressing breast cancer. Nat Commun (2018)
- [2]PMID 21876713 — The Potential Utility of Curcumin in the Treatment of HER-2-Overexpressed Breast Cancer: An In Vitro and In Vivo Comparison Study with Herceptin. Evid Based Complement Alternat Med (2012)
- [3]PMID 212572 — Isolation of two human tumor epithelial cell lines from solid breast carcinomas. J Natl Cancer Inst (1978)
- [4]PMID 94035 — A human breast tumor cell line (BT-474) that supports mouse mammary tumor virus replication. In Vitro (1979)
Disclaimer: RUO; IACUC required. Optimize by strain and pilot. Red tags mark weak or non-metastatic parental endpoints.