Use cases
#4 fat-pad ER+ MCF-7 typically needs exogenous estrogen. This SOP uses Dall: 1×10⁶ in PBS + GFR Matrigel 1:1. Shafie: 2×10⁶ takes in intact nude fat pads; OVX fails even at 20×10⁶. Watch the fat-pad JoVE—not a substitute for surgical training. Do not copy MDA-MB-231’s 2×10⁶ / 100 μL / 25% matrix.
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
Use this page for local orthotopic growth / endocrine efficacy. Kang s.c. 6×10⁶ and Gutierrez tamoxifen-resistance 5×10⁶ are not this SOP’s default.
| Use | Fit | Primary readout | Notes |
|---|---|---|---|
| ER+ orthotopic growth / endocrine efficacy | Recommended | Orthotopic volume, weight, pathology[1][3] | #4 pad; Dall 1×10⁶ + Matrigel 1:1; ovary-intact + lower-dose pellet[1]. |
| Tamoxifen-resistance model | Suitable | Switch endocrine regimen after take; pathway markers[4] | Gutierrez: OVX nude + 0.25 mg-class pellet + fat-pad 5×10⁶. That is a different SOP—not this page’s Dall 1×10⁶. Drug micrograms come only from the IACUC SOP. |
| Flank caliper / tail-vein mets | Not recommended | — | s.c. is the other protocol on this line. Spontaneous distant mets are not the parental MCF-7 default readout. |
Does the workflow match?
Orthotopic endocrine efficacy shares this inoculum skeleton; resistance models change cell number and host plan.
| Use | vs foolproof SOP | Differences vs core SOP |
|---|---|---|
| ER+ orthotopic efficacy | Same core SOP | This page’s Dall 1×10⁶. |
| Tamoxifen resistance | Core + add-ons | Gutierrez uses 5×10⁶ + OVX/pellet; switch drugs after enrollment—not a different needle. |
| s.c. CDX | Other route | Kang 6×10⁶ in Matrigel, not the fat pad. |
Host spec: sex × strain × estrogen
This SOP defaults to female nude #4 pad. Ovary-intact vs OVX must be written with the pellet in the IACUC SOP. Do not mix in MDA-MB-231 orthotopic cell numbers. RUO.
| Indication / context | Sex | Strain / host | Notes |
|---|---|---|---|
| This SOP default: orthotopic growth / endocrine efficacy | Female (required) | Nude nu/nu; ovary-intact + IACUC-sized pellet (Dall) | 1×10⁶ in 1:1 GFR Matrigel, #4 pad[1]. 0.72 mg-class IRA pellets can cause urosepsis; Dall used 0.3–0.5 mg silastic pellets. One week of supplement is not enough. |
| OVX + pellet (classic endocrine model) | Female | Nude; ovariectomized | Shafie: OVX without a pellet fails even at 20×10⁶; an interscapular pellet on inoculum day restores take[2]. Osborne: OVX without estrogen does not take; intact-female growth is often minimal[3]. |
| Tamoxifen-resistance follow-up | Female | OVX nude + 0.25 mg-class pellet (Gutierrez) | Fat-pad 5×10⁶[4]. Do not treat that cell number as Dall’s default. |
| Male | Not recommended | — | Fat-pad models are female-standard. |
In one line
IACUC + estrogen plan → expand → #4 fat-pad 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 (约 1×10⁶ / PBS + GFR Matrigel 1:1（Dall）；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 immunodeficient quarantine
- STR/mycoplasma OK; expand MCF-7
- Implant estradiol pellet per SOP (dorsal; before or on inoculum day)
- Count (≥90% viable)
- PBS + GFR Matrigel 1:1; ~1×10⁶ (Dall)
- Anesthetize; expose #4 pad per JoVE; slow bleb
- Close and warm; weekly caliper + weight
- Optional endocrine therapy at set volume
- Endpoint primary 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 5e+6 规划; dead-space ×1.5. 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 | 14 |
| 10 | 15 |
Inoculum recipe
This SOP dose string: 约 1×10⁶ / PBS + GFR Matrigel 1:1（Dall）；体积按批件，非默认 100 μL. Batch ×1.5 for dead space; load one syringe per animal.[1]
| Item | This SOP | Notes |
|---|---|---|
| Cells / volume | 约 1×10⁶ / PBS + GFR Matrigel 1:1（Dall）；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
#4 mammary fat pad—not flank s.c. Watch this line’s JoVE/paper. Anesthetic: compound card/PMID; IACUC may override.
| Check | Pass | If fail |
|---|---|---|
| Landmark | #4 nipple / fat pad | s.c. bleb: log as failed |
| Push | Slow local bleb; control leak | Immediate leak: log as failed |
| 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 | Orthotopic volume, weight, urinary signs, pathology; optional hormone biomarkers. |
| 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 stocks to a conservative 5×10⁶ (Gutierrez ceiling); this SOP injects Dall’s 1×10⁶. JoVE is the surgery video. Price 1990 is MDA-MB-231/435 and is not listed.
| Paper | Year | Journal | How to use |
|---|---|---|---|
| Dall 2015[1] | Nude / NOD-SCID, #4 pad, ovary-intact | 1×10⁶ PBS + GFR Matrigel 1:1 | 0.3–0.5 mg silastic pellets support take; 0.72 mg IRA can cause urosepsis; one week is not enough. No μL stated |
| Shafie 1981[2] | Intact vs OVX BALB/c nude fat pad | 2×10⁶ log-phase; OVX fails even at 20×10⁶ | Interscapular estradiol pellet on inoculum day restores take. No Matrigel stated |
| Osborne 1985[3] | BALB/c nude ± OVX | No inoculum number in the abstract | Estrogen-dependent growth; 0.01–0.5 mg-class pellets are dose-dependent; removal arrests. Not a 231 dose |
| Gutierrez 2005[4] | OVX nude fat pad, tamoxifen-resistance model | 5×10⁶ | 0.25 mg-class pellet. Not this SOP’s default 1×10⁶; drug doses from IACUC only |
| Kocatürk JoVE 2015[5] | NSG etc. fat-pad surgery video | Example 5×10⁵ | Surgery method, not an MCF-7 dose |
| Soule 1973[6] | Line establishment | JNCI | Identity, not an inoculum SOP |
| Osborne 1987[7] | MCF-7 from different sources | Take / hormone response differed | STR required; do not mix lots |
- 01[Ethics] IACUC; female immunodeficient mice. RUO. Dall/JoVE do not give anesthetic mg/%; immobilize per IACUC. Write ulcer, weight, and urinary endpoints (Dall: 0.72 mg-class pellets can cause urosepsis; Kang high-dose pellets can cause bladder stones)[1].
- 02[Hormone] Implant slow-release estradiol before or on inoculum day, dorsal and away from the pad. Size from the IACUC SOP only. Dall: ovary-intact nude/NSG take on 0.3–0.5 mg silastic pellets; 0.72 mg 60-day IRA pellets can cause urosepsis; one week of supplement does not maintain growth[1]. Shafie: OVX without a pellet does not take; an interscapular pellet on inoculum day restores take[2]. Osborne: 0.01–0.5 mg-class pellets show dose-dependent growth; removal often arrests without regression[3].
- 03[Cells] MCF-7 (MC-h129), 85–90% confluence, ≥90% viable. STR the source (Osborne 1987)[7]. This SOP ~1×10⁶ in PBS + GFR Matrigel 1:1 (Dall)[1]. Shafie intact fat pad is 2×10⁶ (no Matrigel stated)[2]. Gutierrez resistance model is 5×10⁶[4]. Do not default to MDA-MB-231’s 2×10⁶, and do not copy Kang’s s.c. 6×10⁶ into the pad. Volume from the IACUC SOP—Dall does not state μL, so do not default to 100 μL. Culture: datasheet first; Dall used DMEM + 10% FBS + insulin 10 μg/mL[1]. Cobblestone epithelia—do not judge confluence by MDA-MB-231’s spindle “looks sparse” rule.
- 04[Surgery] Expose #4 fat pad per JoVE; slow bleb; close and warm[5]. JoVE’s example 5×10⁵ is not the MCF-7 dose.
- 05
- 06[Endpoint] Primary pathology. Spontaneous mets are not a success criterion. Endocrine micrograms come only from the IACUC SOP—do not copy tamoxifen doses from the Gutierrez page.
Reagents / materials
| Item | Role | Conc. / dose |
|---|---|---|
| MCF-7 cells (MC-h129) | Inoculum | 约 1×10⁶ / PBS + GFR Matrigel 1:1（Dall）；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
Orthotopic volume, weight, urinary signs, pathology; optional hormone biomarkers.
References (PubMed)
- [1]PMID 26640593 — Low Dose, Low Cost Estradiol Pellets Can Support MCF-7 Tumour Growth in Nude Mice without Bladder Symptoms. J Cancer (2015)
- [2]PMID 6265682 — Role of hormones in the growth and regression of human breast cancer cells (MCF-7) transplanted into athymic nude mice. J Natl Cancer Inst (1981)
- [3]PMID 3967234 — Effect of estrogens and antiestrogens on growth of human breast cancer cells in athymic nude mice. Cancer Res (1985)
- [4]PMID 15753463 — Molecular changes in tamoxifen-resistant breast cancer: relationship between estrogen receptor, HER-2, and p38 mitogen-activated protein kinase. J Clin Oncol (2005)
- [5]PMID 25742185 — Orthotopic injection of breast cancer cells into the mammary fat pad of mice to study tumor growth. J Vis Exp (2015)
- [6]PMID 4357757 — A human cell line from a pleural effusion derived from a breast carcinoma. J Natl Cancer Inst (1973)
- [7]PMID 3620713 — Biological differences among MCF-7 human breast cancer cell lines from different laboratories. Breast Cancer Res Treat (1987)
Disclaimer: RUO; IACUC required. Optimize by strain and pilot. Red tags mark weak or non-metastatic parental endpoints.