Use cases
Hind / #4 fat-pad ER+ T47D typically needs exogenous estrogen. This SOP uses Holen: 5×10⁵ in 20 μL (50% Matrigel:50% PBS), left and right hind fat pads; E2 pellet 3 days pre. Matsunaga contrast: 5×10⁶ in 100 μL 50% Matrigel, #4 pad, E2 0.5 mg / 60-day 1 day pre. Not Price 1990, and not 231’s 2×10⁶ / 100 μL.
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
Use this page for local orthotopic growth / endocrine efficacy. Lee s.c. and Price 1990 are not this needle.
| Use | Fit | Primary readout | Notes |
|---|---|---|---|
| Hind fat-pad orthotopic (low cell number) | Recommended | Primary volume, optional bone-mets readout[1] | Holen: 5×10⁵ in 20 μL 50% Matrigel:PBS, left and right hind pads; E2 pellet 3 d pre[1]. That paper also studied bone-mets inhibition—bone readout must be written in the IACUC SOP, not a default success criterion. |
| #4 pad higher-cell contrast | Suitable | Primary volume | Matsunaga: 5×10⁶ in 100 μL 50% Matrigel, bilateral #4 pads; E2 0.5 mg / 60-day 1 d pre; NOD/SCID[2]. Not Holen’s default. |
| s.c. CDX | Not recommended | — | Use the s.c. protocol. Do not turn the 20 μL pad recipe into a flank default. |
Does the workflow match?
Fat-pad orthotopic shares this skeleton; s.c. is another path.
| Use | vs foolproof SOP | Differences vs core SOP |
|---|---|---|
| Fat-pad orthotopic | Same core SOP | This page’s Holen 5×10⁵ / 20 μL. |
| s.c. CDX | Other route | Lee 2×10⁶ flank, not 20 μL into the pad. |
Host spec: sex × strain × estrogen
Holen defaults to females + E2 pellet 3 days pre. Matsunaga used NOD/SCID. RUO.
| Indication / context | Sex | Strain / host | Notes |
|---|---|---|---|
| This SOP default: hind-pad orthotopic | Female | Immunodeficient (Holen) | 5×10⁵ in 20 μL 50% Matrigel; E2 3 d pre[1]. |
In one line
IACUC + estrogen → expand → fat-pad 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 (Holen：5×10⁵ / 20 μL 50% Matrigel:PBS。Matsunaga 对照 5×10⁶ / 100 μL 50% 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 quarantine
- Implant E2 pellet per IACUC (Holen: 3 d pre)
- STR OK; expand parental T47D
- 5×10⁵ in 20 μL 50% Matrigel:PBS
- Inject left and right hind fat pads
- Weekly caliper + weight
- Endpoint primary pathology (bone only if written in SOP)
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 5e+5 规划; dead-space ×1.5. Do not reuse MDA-MB-231 orthotopic 2×10⁶ math.
| Animals | T75 flasks |
|---|---|
| 1 | 1 |
| 2 | 1 |
| 3 | 1 |
| 4 | 1 |
| 5 | 1 |
| 6 | 1 |
| 7 | 2 |
| 8 | 2 |
| 9 | 2 |
| 10 | 2 |
Inoculum recipe
This SOP dose string: Holen：5×10⁵ / 20 μL 50% Matrigel:PBS。Matsunaga 对照 5×10⁶ / 100 μL 50% Matrigel. Batch ×1.5 for dead space; load one syringe per animal.[1]
| Item | This SOP | Notes |
|---|---|---|
| Cells / volume | Holen：5×10⁵ / 20 μL 50% Matrigel:PBS。Matsunaga 对照 5×10⁶ / 100 μL 50% 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
#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 (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 | Orthotopic volume, pathology; bone readout only if written in the SOP. |
| 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. Holen/Matsunaga do not give inoculum anesthetic mg/%; immobilize per IACUC. Write weight and estrogen-related endpoints.
- 02
- 03
- 04[Surgery] Slow inject into hind fat pads (Holen) or #4 pads (Matsunaga). Not flank s.c.
- 05[Monitor] Volume ≈ 4/3 π r³ (Holen; r=(L+W)/4)[1]. Do not mix formulas.
- 06[Endpoint] Primary pathology. Bone mets only when the IACUC SOP writes the follow-up plan.
Reagents / materials
| Item | Role | Conc. / dose |
|---|---|---|
| T47D cells (MC-h209) | Inoculum | Holen：5×10⁵ / 20 μL 50% Matrigel:PBS。Matsunaga 对照 5×10⁶ / 100 μL 50% Matrigel |
| Matrigel (commonly used) | Engraftment aid | 常 1:1，按 SOP |
| 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 | Holen/Matsunaga 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
Orthotopic volume, pathology; bone readout only if written in the SOP.
References (PubMed)
- [1]PMID 26585891 — Oestrogen receptor positive breast cancer metastasis to bone: inhibition by targeting the bone microenvironment in vivo. Clin Exp Metastasis (2016)
- [2]PMID 33535554 — Novel Anti-FOLR1 Antibody-Drug Conjugate MORAb-202 in Breast Cancer and Non-Small Cell Lung Cancer Cells. Antibodies (Basel) (2021)
- [3]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.