Use cases
Left-ventricular MDA-MB-231 (or BO/B02) seeds experimental osteolysis. Parental hits bone at 3–4 weeks ± adrenal/ovary/brain; BO is nearly bone-only. Written as a JoVE novice pack—not a substitute for surgical training.
Catalog: MC-h133 · Product modeling page · In-vitro spheroid
RUO. IACUC, SPF, and LV-injection training required. No anesthetic doses here; not a substitute for JoVE or the veterinary SOP. No Matrigel. Do not inject without bright-red flashback.
Campbell JoVE: bone-metastasis models (LV + tibia, video) · PMID 22972196
Use-case overview
Use this page for bone homing/colonization; switch to intratibial for local bone microenvironment, or spontaneous orthotopic mets for the full cascade.
| Use | Fit | Primary readout | Notes |
|---|---|---|---|
| Experimental osteolysis / bone-targeted screen | Recommended | X-ray/μCT, TRAP, hind-limb function[1][2][3] | 1×10⁵ / 100 μL PBS; female nude 4–6 wk. |
| BO vs parental organ tropism | Suitable | Bone vs visceral foci[1] | Same needle; necropsy must log non-bone organs. |
| Local established bone disease or brain as primary readout | Not recommended | This SOP includes homing; use the BR page for brain | Use intratibial for local mechanisms. BR LV belongs on the brain page—not here. |
Does the workflow match?
Osteolysis screens and BO comparisons share this LV skeleton; tibia and brain need another protocol.
| Use | vs foolproof SOP | Differences vs core SOP |
|---|---|---|
| Experimental osteolysis | Same core SOP | This page. |
| BO vs parental | Core + add-ons | Same needle; necropsy spectrum differs. |
| Intratibial / brain / spontaneous | Other route | Marrow, BR LV, or fat pad—not this needle. |
Host spec: sex × strain × age
Yoneda/JoVE default female nude 4–6 wk[1][2]. Wright also lists NOD-SCID/NSG[3]. Males are not the default. RUO.
| Indication / context | Sex | Strain / host | Notes |
|---|---|---|---|
| This SOP default | Female (required) | Nude; 4–6 weeks | 1×10⁵ / 100 μL; pilot n=3–5. |
| Deeper immunodeficiency | Female | NOD-SCID or NSG | Wright lists these strains[3]; burden may arrive faster. |
| Male | Not the default | — | The SOPs above are female. |
In one line
IACUC → prepare cells → left-ventricle inject → X-ray/BLI/clinical signs → bone pathology.
D−1 / D0 timeline
1. D−7 to D−3
Quarantine female nudes; write paraplegia/cachexia endpoints.
2. D−1
Feed at 80–90% confluence[[1]][[2]].
3. D0
Ice PBS 1×10⁵/100 μL → LV. Finish in 30 min.
4. From D+14
Weight, hind limbs; X-ray ~week 3.
Protocol overview
Scenario-specific flowchart (core engraftment skeleton with host/therapy or imaging/readout changes).
- IACUC + immunodeficient host (paraplegia/cachexia endpoints)
- Expand parental or BO/B02 (log passage; T75 on expansion table)
- Single-cell suspension (no Matrigel)
- Anesthetize; LV inject (bright-red flashback, 28G, 100 μL)
- X-ray / BLI / weight
- Endpoint long-bone pathology
Novice pack (literature cases)
Flowchart buttons open the matching table. Full pack below, in use order.
Unpack list (LV bone mets, plan per mouse)
Arterial dissemination—no Matrigel. Watch the Campbell JoVE video. No anesthetic doses on this page.[1][2]
| Item | Per mouse / study | Notes |
|---|---|---|
| MDA-MB-231 or BO/B02 subline MC-h133 | Start from 1 vial | STR/mycoplasma-qualified; BO is more bone-restricted than parental[1] |
| FBS MC100 | 10% complete medium | Prefer regular grade |
| Medium | ~15 mL per T75 | Datasheet first; JoVE splits at 80–90% confluence[2] |
| Ice-cold Ca²⁺/Mg²⁺-free PBS | 100 μL/mouse | JoVE: ice PBS prevents clumping/emboli; finish in 30 min[2] |
| Trypsin-EDTA | ~3–5 mL per T75 | JoVE uses dilute trypsin for fast detach, fewer clumps[2] |
| T75 | See expansion table; JoVE ~1 flask / 8–10 mice[2] | Harvest at 80–90% confluence |
| 300 μL insulin syringe 28G ½″ | 1 per mouse | Yoneda 28G; JoVE uses a hub air gap to see the pulse—do not inject air into the ventricle[1][2] |
| X-ray / μCT / optional GFP imaging | Shared | Osteolysis often not radiographic until ~week 3; day-14 GFP often ex vivo[2] |
Expansion cases: mice vs T75 (LV 1×10⁵)
1×10⁵/mouse + 20% (insulin dead space is smaller than tuberculin). Log-phase T75 at 5×10⁶. JoVE: 1 flask covers 8–10 mice[2]. Do not reuse orthotopic 2×10⁶ flask math.
| Mice | T75 flasks |
|---|---|
| 1 | 1 |
| 2 | 1 |
| 3 | 1 |
| 4 | 1 |
| 5 | 1 |
| 6 | 1 |
| 7 | 1 |
| 8 | 1 |
| 9 | 1 |
| 10 | 1 |
Inoculum recipe (LV; no matrix)
All three papers use ~1×10⁵ in 100 μL PBS. BO/B02 same dose, narrower organ spectrum.[1][2][3]
| Item | This SOP | Literature |
|---|---|---|
| Cells | 1×10⁵ / mouse | Yoneda / JoVE / Wright all 1×10⁵[1][2][3] |
| Volume / vehicle | 100 μL ice-cold PBS | No Matrigel; ice; finish in 30 min[2] |
| Density | 1×10⁶ / mL | JoVE resuspends at 10⁶/mL and injects 100 μL[2] |
| Batch (10 mice) | ~1.2×10⁶ → 1 T75 | JoVE: 1 T75 / 8–10 mice[2] |
| Viability | ≥90% singles | Clumps → acute brain/lung emboli |
Injection checklist (left ventricle, not tail vein or tibia)
Watch JoVE. Do not inject without a bright-red pulsatile flashback. No anesthetic doses here.[1][2]
| Check | Pass | If fail |
|---|---|---|
| Landmark | Midway sternal notch–xiphoid, slightly anatomical left; 4th intercostal[2] | Unclear: stop; use JoVE Fig. 1 |
| Flashback | Bright-red arterial pulse into the hub[1][2] | Dark/no pulse: likely RV or chest—withdraw, do not inject |
| Push | Slow 100 μL with the needle still, to avoid puncture or chest spill[2] | High resistance or collapse: stop; humane endpoint |
| Air gap | Hub air is only to visualize the pulse—never inject air[2] | If air is pushed: treat as embolus / endpoint |
| Recover | Warm until righting; analgesia per IACUC | Prolonged recovery or distress → vet SOP |
Culture card (MDA-MB-231 / MC-h133)
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.
Yoneda: feed 24 h before harvest at subconfluence[1]. JoVE: 80–90% confluence; rounding means overcrowding—do not inject[2].
| Item | Practice |
|---|---|
| Medium | Datasheet first. JoVE: do not overcrowd; keep the fibroblastic/pseudopod morphology[2]. |
| Confluence | Harvest at 80–90% (same idea as the 85–90% schematic). Overconfluence drops in-vivo metastatic potential[2]. |
| Day −1 | Feed[1]; one T75 often covers 8–10 mice. |
| Subline | Log parental, BO, and BR separately; do not mix organ expectations[1]. |
What success looks like (expected, not a guarantee)
Parental: bone at 3–4 weeks ± occasional adrenal/ovary/brain; BO almost bone-only.[1][2]
| Time point | Typical appearance |
|---|---|
| Day 0 | Bright-red flashback then inject; mouse active after recovery |
| ~Week 2 | GFP foci often only ex vivo; radiographs often still negative[2] |
| ~Weeks 3–4 | Osteolysis visible; JoVE sacrifice ~week 4. Cachexia/paraplegia often days 21–35[2] |
| Take | Bone-seeking lines can reach ~90% long-bone take after LV[3]; parental also seeds viscera—log it |
Troubleshooting
Paralysis and cachexia follow the IACUC SOP immediately—do not delay for a radiograph.
| Sign | Likely cause | Action |
|---|---|---|
| Immediate death / lung bleed | RV, chest spill, air, or clumps | Stop; audit needle plane and mix |
| Injected without red flashback | Missed LV | Log as failed; review JoVE |
| No osteolysis by week 4 | Missed inject, dead cells, wrong subline | Audit flashback log; do not blindly raise dose |
| Paraplegia / sharp weight loss | Spine burden | Humane endpoint now |
Monitoring log fields
Weekly weight, hind limbs, X-ray. No caliper volumes on this page.
| Field | How to log |
|---|---|
| Date / D0 | LV day |
| Bright pulsatile flashback | Y/N (N = failed inject) |
| Weight / hind limb / paraplegia | Stop if endpoint hit |
| Radiographic osteolysis | Distal femur / proximal tibia typical[3] |
Literature case comparison
This SOP uses 1×10⁵ / 100 μL. Open the PMID for the JoVE video.
| Paper | Host | Cells | Method | Readout |
|---|---|---|---|---|
| Yoneda 2001[1] | ♀ nude 4 wk | 1×10⁵ / 0.1 mL PBS; 28G | Prone; LV after bright arterial return | Parental bone ± adrenal/ovary/brain at 3–4 wk; BO bone-only, BR brain-only |
| Campbell JoVE 2012[2] | ♀ nude 4–6 wk | 1×10⁶/mL → 100 μL; bone-tropic GFP clone | 28G insulin; mid notch–xiphoid, left | Osteolysis ~wk 3; cachexia/paraplegia d21–35; watch the video |
| Wright 2016[3] | Nude / NOD-SCID / NSG | 1×10⁵ LV | Arterial spread to long bones, spine, jaw | Osteolysis 2–3 wk; sublines up to ~90% |
| Peyruchaud 2001[4] | Nude | 1×10⁵ / 0.1 mL | LV; B02 selection | GFP early osteolysis; do not mix B02 tail-vein SOP onto this LV page |
- 01[Ethics] IACUC; LV training. Paraplegia/cachexia endpoints. RUO. Watch Campbell JoVE[2]. No anesthetic doses here.
Arterial dissemination—no Matrigel. Watch the Campbell JoVE video. No anesthetic doses on this page.[1][2]
Item Per mouse / study Notes MDA-MB-231 or BO/B02 subline MC-h133 Start from 1 vial STR/mycoplasma-qualified; BO is more bone-restricted than parental[1] FBS MC100 10% complete medium Prefer regular grade Medium ~15 mL per T75 Datasheet first; JoVE splits at 80–90% confluence[2] Ice-cold Ca²⁺/Mg²⁺-free PBS 100 μL/mouse JoVE: ice PBS prevents clumping/emboli; finish in 30 min[2] Trypsin-EDTA ~3–5 mL per T75 JoVE uses dilute trypsin for fast detach, fewer clumps[2] T75 See expansion table; JoVE ~1 flask / 8–10 mice[2] Harvest at 80–90% confluence 300 μL insulin syringe 28G ½″ 1 per mouse Yoneda 28G; JoVE uses a hub air gap to see the pulse—do not inject air into the ventricle[1][2] X-ray / μCT / optional GFP imaging Shared Osteolysis often not radiographic until ~week 3; day-14 GFP often ex vivo[2] - 02
- 03
- 04[Monitor] Weight, hind limbs; X-ray ~week 3. Day-14 GFP is often ex vivo[2].
Weekly weight, hind limbs, X-ray. No caliper volumes on this page.
Field How to log Date / D0 LV day Bright pulsatile flashback Y/N (N = failed inject) Weight / hind limb / paraplegia Stop if endpoint hit Radiographic osteolysis Distal femur / proximal tibia typical[3] Paralysis and cachexia follow the IACUC SOP immediately—do not delay for a radiograph.
Sign Likely cause Action Immediate death / lung bleed RV, chest spill, air, or clumps Stop; audit needle plane and mix Injected without red flashback Missed LV Log as failed; review JoVE No osteolysis by week 4 Missed inject, dead cells, wrong subline Audit flashback log; do not blindly raise dose Paraplegia / sharp weight loss Spine burden Humane endpoint now - 05[Endpoint] Femur/tibia/spine X-ray, HE, TRAP; log viscera for parental[1].
Weekly weight, hind limbs, X-ray. No caliper volumes on this page.
Field How to log Date / D0 LV day Bright pulsatile flashback Y/N (N = failed inject) Weight / hind limb / paraplegia Stop if endpoint hit Radiographic osteolysis Distal femur / proximal tibia typical[3] Paralysis and cachexia follow the IACUC SOP immediately—do not delay for a radiograph.
Sign Likely cause Action Immediate death / lung bleed RV, chest spill, air, or clumps Stop; audit needle plane and mix Injected without red flashback Missed LV Log as failed; review JoVE No osteolysis by week 4 Missed inject, dead cells, wrong subline Audit flashback log; do not blindly raise dose Paraplegia / sharp weight loss Spine burden Humane endpoint now - 06[Limits] Skips intravasation. Use intratibial for local bone mechanisms; the BR page for brain.
- 07[QC] Flashback log; independent repeats. Troubleshoot from the checklist first.
Reagents / materials
| Item | Role | Conc. / dose |
|---|---|---|
| MDA-MB-231 cells (MC-h133) | LV inoculum | 1×10⁵ / 100 μL 冰 PBS[[1]][[2]] |
| Bone-seeking subline (optional, BO/B02) | Enrich bone, reduce viscera | 同 1×10⁵；来源与代次必须记录[[1]][[4]] |
| Anesthetic / analgesic | Surgery & welfare | 按伦理批件与兽医 SOP |
| Test drug / vehicle (optional) | Treatment arm | 按药理方案 |
Readouts
Osteolytic scores, μCT/X-ray, BLI/GFP, bone pathology, weight/mobility.
References (PubMed)
- [1]PMID 11499871 — A bone-seeking clone exhibits different biological properties from the MDA-MB-231 parental human breast cancer cells and a brain-seeking clone in vivo and in vitro. J Bone Miner Res (2001)
- [2]PMID 22972196 — Models of bone metastasis. J Vis Exp (2012)
- [3]PMID 27867497 — Murine models of breast cancer bone metastasis. Bonekey Rep (2016)
- [4]PMID 11697798 — Early detection of bone metastases in a murine model using fluorescent human breast cancer cells: application to the use of the bisphosphonate zoledronic acid in the treatment of osteolytic lesions. J Bone Miner Res (2001)
Disclaimer: RUO; IACUC required. Optimize by strain and pilot. Red tags mark weak or non-metastatic parental endpoints.