Use cases
ER+/PR+ T47D flank s.c. CDX usually needs exogenous estrogen for endocrine pharmacology. This SOP uses Lee: 2×10⁶ flank s.c.; 60-day estrogen pellet 7 days pre—μL/gel not stated. Lee’s other arm 1×10⁷ is for established-tumor therapy, not the default inoculum. Not fat-pad orthotopic. Do not copy MDA-MB-231’s 2×10⁶ / 100 μL or Price 1990.
Catalog: MC-h209 · 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. Fat-pad orthotopic is another protocol. Price 1990 is MDA-MB-231/435—not T47D.
| Use | Fit | Primary readout | Notes |
|---|---|---|---|
| ER+ endocrine s.c. screen | Recommended | s.c. volume, weight, pathology[1] | Lee: ♀ nude 8 wk, 2×10⁶ flank; E2 60-day pellet 7 d pre; tumors within ~3 weeks[1]. μL/gel not stated. |
| High-cell established-tumor contrast | Suitable | Intratumoral therapy after ~6–7 mm | Lee: 1×10⁷ flank; ~6–7 mm by day 10 then intratumoral adenovirus—not the default inoculum[1]. |
| Mammary fat-pad orthotopic | Not recommended | — | Use the orthotopic protocol. Do not copy Lee’s s.c. 2×10⁶ into the fat pad, and do not default s.c. to Holen’s 5×10⁵ / 20 μL. |
Does the workflow match?
Endocrine screens share this s.c. skeleton; the fat pad is a different protocol.
| Use | vs foolproof SOP | Differences vs core SOP |
|---|---|---|
| ER+ s.c. screen | Same core SOP | This page’s Lee 2×10⁶. |
| Fat-pad orthotopic | Other route | Holen 5×10⁵ / 20 μL or Matsunaga 5×10⁶ / 100 μL, not the s.c. default. |
Host spec: sex × strain × estrogen
Human T47D uses immunodeficient females + exogenous estrogen. RUO.
| Indication / context | Sex | Strain / host | Notes |
|---|---|---|---|
| This SOP default: s.c. endocrine screen | Female | Nude (Lee) | 2×10⁶ flank; E2 60-day pellet 7 d pre[1]. |
| Male | Not recommended | — | ER+ mammary models are female-standard. |
In one line
IACUC + estrogen plan → expand → s.c. inject → 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 (Lee：2×10⁶ 皮下（μL/胶未写）。1×10⁷ 臂仅已成瘤干预对照).
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 T47D
- Implant E2 pellet per IACUC (Lee: 7 d pre)
- Count (≥90% viable)
- ~2×10⁶ / mouse (Lee; volume from IACUC)
- 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-h209 datasheet for medium. Matrigel only if the compound card lists it. Anesthetic: compound card/PMID; IACUC may override.[1]
| Item | Per mouse / study | Notes |
|---|---|---|
| T47D cells MC-h209 | 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) | Lee: 60-day pellet, 7 d pre. Holen: 3 d pre. Matsunaga: 0.5 mg / 60-day, 1 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+6 规划; dead-space ×1.2. Do not reuse MDA-MB-231 orthotopic 2×10⁶ math.
| Animals | T75 flasks |
|---|---|
| 1 | 1 |
| 2 | 1 |
| 3 | 2 |
| 4 | 2 |
| 5 | 3 |
| 6 | 3 |
| 7 | 4 |
| 8 | 4 |
| 9 | 5 |
| 10 | 5 |
Inoculum recipe
This SOP dose string: Lee：2×10⁶ 皮下（μL/胶未写）。1×10⁷ 臂仅已成瘤干预对照. Batch ×1.2 for dead space; load one syringe per animal.[1]
| Item | This SOP | Notes |
|---|---|---|
| Cells / volume | Lee：2×10⁶ 皮下（μL/胶未写）。1×10⁷ 臂仅已成瘤干预对照 | 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 (T47D / MC-h209)
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-h209). 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
- 01[Ethics] IACUC; female immunodeficient mice. RUO. Lee s.c. does not give anesthetic mg/%; immobilize per IACUC. Write ulcer, weight, and estrogen-related humane endpoints.
- 02[Hormone] Implant slow-release estradiol before inoculum (size from the IACUC SOP only). Lee: 60-day pellet, 7 d pre[1].
- 03[Cells] T47D (MC-h209), STR/mycoplasma-qualified. Keydar: human breast-carcinoma origin[2]. ER+/PR+—usually needs exogenous estrogen. Log phase, ≥90% viable. Datasheet first.
- 04[Suspension] This SOP ~2×10⁶ / mouse (Lee)[1]. μL/gel not stated—do not default to 100 μL or MDA-MB-231’s gel recipe. Lee’s 1×10⁷ arm is established-tumor therapy, not the default inoculum. Do not treat Price 1990 as a T47D dose.
- 05[Inject] Flank s.c. bleb; avoid muscle. Keep the pellet away from the inject site.
This SOP dose string: Lee：2×10⁶ 皮下（μL/胶未写）。1×10⁷ 臂仅已成瘤干预对照. Batch ×1.2 for dead space; load one syringe per animal.[1]
Item This SOP Notes Cells / volume Lee：2×10⁶ 皮下（μL/胶未写）。1×10⁷ 臂仅已成瘤干预对照 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 formula from the IACUC SOP; Lee used L×W×D×π/6[1].
- 07[Endpoint] Tumor pathology. Bone mets are not an s.c. success criterion.
Reagents / materials
| Item | Role | Conc. / dose |
|---|---|---|
| T47D cells (MC-h209) | Inoculum | Lee：2×10⁶ 皮下（μL/胶未写）。1×10⁷ 臂仅已成瘤干预对照 |
| Matrigel (not stated for this SOP) | Engraftment aid | Lee 未写。仅当改走 Matsunaga/Holen 脂肪垫配方时按该文混合 |
| Exogenous estrogen (slow-release pellet / IACUC size) | Support ER+/PR+ T47D engraftment | 规格只来自 IACUC。文献对照：Lee 60 天丸，接种前 7 天；Holen 接种前 3 天植入；Matsunaga 0.5 mg / 60 天，接种前 1 天。不要把网页毫克数当唯一正确。 |
| Anesthetic / analgesic | Surgery & welfare | Lee 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 11788792 — Adenovirus-directed expression of dominant negative estrogen receptor induces apoptosis in breast cancer cells and regression of tumors in nude mice. Mol Med (2001)
- [2]PMID 228940 — Establishment and characterization of a cell line of human breast carcinoma origin. Eur J Cancer (1979)
Disclaimer: RUO; IACUC required. Optimize by strain and pilot. Red tags mark weak or non-metastatic parental endpoints.