Use cases
ER+ MCF-7 flank/forearm s.c. growth usually needs exogenous estrogen and Matrigel for endocrine pharmacology. Kang: 6×10⁶ in ice-cold Matrigel. Not fat-pad orthotopic and not a mets model. Do not copy MDA-MB-231’s 2×10⁶ / 100 μL.
Catalog: MC-h129 · 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 and pellet size live on the orthotopic protocol. Do not copy Price 1990 MDA-MB-231/435 numbers.
| Use | Fit | Primary readout | Notes |
|---|---|---|---|
| ER+ endocrine / antiestrogen screen | Recommended | s.c. volume, weight, pathology[1][2] | Kang: OVX nude + estradiol pellet + 6×10⁶ in Matrigel, forearm s.c.[1]. |
| Mammary fat-pad orthotopic | Not recommended | — | Use the orthotopic protocol. Do not copy Kang’s s.c. 6×10⁶ into the fat pad, and do not default s.c. to Dall’s orthotopic 1×10⁶. |
| Spontaneous distant mets | Not recommended | — | Parental MCF-7 s.c. is not a metastasis model. |
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. |
| Fat-pad orthotopic | Other route | #4 pad + Dall 1×10⁶, not Kang s.c. 6×10⁶. |
Host spec: sex × strain × estrogen
Human MCF-7 uses immunodeficient females. OVX vs ovary-intact must be written with pellet size in the IACUC SOP. Kang used OVX + pellet; high-dose pellets raised deaths. RUO.
| Indication / context | Sex | Strain / host | Notes |
|---|---|---|---|
| This SOP default: s.c. endocrine screen | Female | BALB/c nude (nu/nu); Kang used OVX + pellet | 6×10⁶ in ice-cold Matrigel, forearm s.c.[1]. Pellet size from the IACUC SOP; see Kang low-dose vs 1.7 mg-class mortality. |
| Ovary-intact + lower-dose pellet | Female | Nude or NOD-SCID (see Dall orthotopic paper) | Dall is fat-pad, not s.c.—use it for hormone-toxicity discussion, not the cell number. |
| Male | Not recommended | — | ER+ mammary 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 (约 6×10⁶ / 冰 Matrigel（Kang）；体积按批件，非默认 100 μL).
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 (OVX per SOP)
- STR/mycoplasma OK; expand MCF-7 (log source and passage)
- Implant estradiol pellet per IACUC (dorsal, away from inject site)
- Count (≥90% viable)
- Ice-cold Matrigel mix; ~6×10⁶ / mouse (Kang)
- Flank or forearm s.c. bleb
- Weekly caliper + weight; log urinary signs / sharp weight drop
- 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-h129 datasheet for medium. Matrigel only if the compound card lists it. Anesthetic: compound card/PMID; IACUC may override.[1]
| Item | Per mouse / study | Notes |
|---|---|---|
| MCF-7 cells MC-h129 | 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 or on inoculum day (dorsal flank, away from the inject site) | Dall: ovary-intact + lower-dose pellets can take; 0.72 mg-class IRA pellets can cause urosepsis. Kang: 1.7 mg 60-day pellets raised mortality (bladder stones/renal injury). One week of supplement is not enough (Dall). 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 6e+6 规划; dead-space ×1.2. Do not reuse MDA-MB-231 orthotopic 2×10⁶ math.
| Animals | T75 flasks |
|---|---|
| 1 | 2 |
| 2 | 3 |
| 3 | 5 |
| 4 | 6 |
| 5 | 8 |
| 6 | 9 |
| 7 | 11 |
| 8 | 12 |
| 9 | 13 |
| 10 | 15 |
Inoculum recipe
This SOP dose string: 约 6×10⁶ / 冰 Matrigel（Kang）；体积按批件，非默认 100 μL. Batch ×1.2 for dead space; load one syringe per animal.[1]
| Item | This SOP | Notes |
|---|---|---|
| Cells / volume | 约 6×10⁶ / 冰 Matrigel（Kang）；体积按批件，非默认 100 μL | 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 (MCF-7 / MC-h129)
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-h129). 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, urinary signs, 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
T75 is planned to Kang’s 6×10⁶. s.c. is not orthotopic. Pellet milligrams are for reading the paper—not a default dose.
| Paper | Year | Journal | How to use |
|---|---|---|---|
| Kang 2009[1] | OVX BALB/c nude, forearm s.c. | 6×10⁶ in ice-cold Matrigel | Contrast: 1.7 mg-class 60-day pellets raised deaths; 0.18 mg-class still supported tumors. No μL stated |
| Osborne 1985[2] | BALB/c nude | No cell number in the abstract | OVX without estrogen does not take; intact growth is weak; pellet removal arrests without reliable regression |
| Soule 1973[3] | Line establishment | JNCI | Identity / culture background—not an inoculum SOP |
| Osborne 1987[4] | MCF-7 lots from different labs | Nude take differed | STR required; the ATCC-named line then was from a different patient |
| Dall 2015[5] | Contrast: fat-pad orthotopic | 1×10⁶ + Matrigel 1:1 | Not an s.c. dose; use for pellet urinary-toxicity contrast |
- 01[Ethics] IACUC; female immunodeficient mice. RUO. Kang/Osborne do not give anesthetic mg/%; immobilize per IACUC. Write ulcer, weight, and urinary humane endpoints (high-dose estrogen can cause bladder stones/renal injury)[1].
- 02[Hormone] Implant slow-release estradiol before or on inoculum day (size from the IACUC SOP only). Kang: ~40% 28-day survival on 1.7 mg-class 60-day pellets vs ~80% on 0.18 mg-class, and the low dose still supported tumors[1]. Osborne: OVX without estrogen does not take; intact nude circulating estrogen is low and growth is often minimal[2].
- 03[Cells] MCF-7 (MC-h129), STR/mycoplasma-qualified. Osborne 1987: MCF-7 lots differ in take and hormone response; the ATCC-named line then was from a different patient—document the source[4]. Log phase, ≥90% viable. Datasheet first; Kang used MEM + 10% FBS[1]. Cellosaurus doubling is multi-source ~24–80 h (DSMZ ~50 h, range 30–72 h)—not a measured lot.
- 04[Suspension] ~6×10⁶ in ice-cold Matrigel (Kang forearm s.c.)[1]. Volume from the IACUC SOP—do not default to 100 μL, Dall’s orthotopic 1×10⁶, or MDA-MB-231’s 2×10⁶.
- 05[Inject] Flank or forearm s.c. bleb; avoid muscle. Keep the pellet away from the inject site.
- 06[Monitor] Volume ≈ L×W²/2 (this site’s log). Kang used L×W×T×0.524—do not mix formulas[1]. Log weight and urinary irritation.
- 07[Endpoint] Tumor pathology. Pellet removal often arrests growth without regression[2]. Mets are not a success criterion.
Reagents / materials
| Item | Role | Conc. / dose |
|---|---|---|
| MCF-7 cells (MC-h129) | Inoculum | 约 6×10⁶ / 冰 Matrigel（Kang）；体积按批件，非默认 100 μL |
| Matrigel (commonly used) | Engraftment aid | 常 1:1，按 SOP |
| Exogenous estrogen (slow-release pellet / IACUC size) | Support ER+ engraftment | 规格只来自 IACUC。文献对照：Dall 完整卵巢+0.3–0.5 mg 硅胶丸；Kang 0.18 mg 级仍可支持皮下成瘤。不要把网页毫克数当唯一正确。 |
| Anesthetic / analgesic | Surgery & welfare | 文献有 mg/% 则写在该协议化合物卡；批件可覆盖 |
| Test drug / vehicle (optional) | Treatment arm | 按药理方案 |
Readouts
s.c. tumor volume/weight, body weight, urinary signs, pathology.
References (PubMed)
- [1]PMID 19122299 — Low dose estrogen supplementation reduces mortality of mice in estrogen-dependent human tumor xenograft model. Biol Pharm Bull (2009)
- [2]PMID 3967234 — Effect of estrogens and antiestrogens on growth of human breast cancer cells in athymic nude mice. Cancer Res (1985)
- [3]PMID 4357757 — A human cell line from a pleural effusion derived from a breast carcinoma. J Natl Cancer Inst (1973)
- [4]PMID 3620713 — Biological differences among MCF-7 human breast cancer cell lines from different laboratories. Breast Cancer Res Treat (1987)
- [5]PMID 26640593 — Low Dose, Low Cost Estradiol Pellets Can Support MCF-7 Tumour Growth in Nude Mice without Bladder Symptoms. J Cancer (2015)
Disclaimer: RUO; IACUC required. Optimize by strain and pilot. Red tags mark weak or non-metastatic parental endpoints.