Use cases
Tail-vein MDA-MB-231 (often Luc-labeled) bypasses primary growth/intravasation to score circulating survival, extravasation, and lung colonization. Lung-tropic LM2 optional. Written as a novice pack—not a substitute for IACUC or injection training.
Catalog: MC-h133 · Product modeling page · In-vitro spheroid
Research use only (RUO)—not clinical care. IACUC approval and SPF housing are required. This page does not give anesthetic/analgesic doses and does not replace the veterinary SOP. Do not proceed without approval. Tail vein is not flank s.c. and not #4 fat pad; no Matrigel in the inoculum.
Use-case overview
Use this page for lung colonization; switch to spontaneous orthotopic mets for the full cascade, or intracardiac/brain protocols for bone/brain. Do not reuse flank or fat-pad SOPs i.v.
| Use | Fit | Primary readout | Notes |
|---|---|---|---|
| Experimental lung colonization / anti-met screen | Recommended | Lung foci / BLI / HE[1][2][3] | Parental often 1×10⁶ / 100 μL PBS; LM2 at a lower dose. |
| LM2 vs parental lung tropism | Suitable | BLI/lung burden at a matched cell number[1] | Same inject skeleton; do not give LM2 the parental 1×10⁶. |
| Full cascade (intravasation) or bone/brain as primary readout | Not recommended | This SOP skips the primary and intravasation; bone/brain are not the target organs | Use spontaneous orthotopic mets, or intracardiac/brain protocols. Zhou rarely saw muscle/bone after tail vein (1/17)—not a bone model[3]. |
Does the workflow match?
Lung colonization and LM2 comparisons share this i.v. skeleton; spontaneous mets and bone/brain need another protocol.
| Use | vs foolproof SOP | Differences vs core SOP |
|---|---|---|
| Experimental lung colonization / anti-met screen | Same core SOP | This page is the core SOP. |
| LM2 vs parental | Core + add-ons | Same needle; lower LM2 dose and shorter follow-up. |
| Spontaneous mets / LV bone / brain | Other route | Fat pad, left ventricle, or intracranial—not tail vein. |
Host spec: sex × strain × age
Minn used female nudes 4–6 wk[1], Yang NOD-SCID 4–6 wk[2], Ring NSG 8–10 wk[4]. This SOP defaults to female nudes. NSG lung burden arrives faster—watch endpoints earlier. Quarantine ≥3–7 days. RUO.
| Indication / context | Sex | Strain / host | Notes |
|---|---|---|---|
| This SOP default: parental lung colonization | Female (required) | Nude nu/nu; 4–6 weeks; quarantine ≥3–7 days | Matches Minn/Zhou. Parental ~1×10⁶ / 100 μL; 2×10⁵ is mostly micromets[1][3]. Pilot n=3–5. |
| Luc BLI methods | Female | NOD-SCID; 4–6 weeks | Yang: heat dilation, 100 μL, 26G tuberculin[2]. |
| Faster lung burden on NSG | Female | NSG; 8–10 weeks | Ring: 1×10⁶ / 100 μL medium; controls large by ~5 wk, endpoint ~day 57[4]. |
| Male | Not the default | — | The SOPs above are female; males need written justification. |
In one line
IACUC → prepare Luc/parental or LM2 → tail-vein inject → BLI/lung burden → endpoint lung pathology.
D−1 / D0 timeline
1. D−7 to D−3
SPF quarantine; confirm female/age; write respiratory-distress endpoints; IACUC in hand.
2. D−1
Feed cells at 85–90% confluence; weigh; confirm the reporter is still on Luc lines.
3. D0 morning
PBS wash → trypsin → neutralize/spin → PBS resuspend → count (≥90% viable, no clumps).
4. D0 mix
Adjust in PBS to 100 μL/mouse (+ ~50% extra) per the recipe card. No Matrigel.
5. D0 inject
Warm → restrain → 26G lateral vein, 100 μL. No s.c. bleb.
6. D0 after inject
Watch breathing; Luc: immediate BLI to drop failed injects[[1]]. Log dose, subline, and passage.
7. From D+3
Weight + breathing; scheduled BLI for Luc. Stop at humane endpoints.
Protocol overview
Scenario-specific flowchart (core engraftment skeleton with host/therapy or imaging/readout changes).
- IACUC + immunodeficient quarantine (respiratory endpoints)
- Expand MDA-MB-231 ± Luc/LM2 (log passage; T75 on expansion table)
- Single-cell count (viability ≥90%, no Matrigel)
- Tail-vein inject (warm, 26G, 100 μL)
- BLI / weight / breathing
- Endpoint lung pathology
Novice pack (literature cases)
Flowchart buttons open the matching table. Full pack below, in use order.
Unpack list (tail-vein lung colonization, plan per mouse)
SKUs link to catalog items. This route is a single-cell i.v. suspension—no Matrigel (pulmonary emboli). Follow the MC-h133 datasheet.[1][2][3]
| Item | Per mouse / study | Notes |
|---|---|---|
| MDA-MB-231 or LM2 subline MC-h133 | Start from 1 vial | STR / mycoplasma-qualified; log passage; LM2 colonizes lung far better than parental[1] |
| FBS (routine culture) MC100 | 10% in complete medium | Prefer regular grade |
| Medium (DMEM or RPMI) | ~15 mL per T75 | Minn: high-glucose DMEM + 10% FBS[1]; Zhou: RPMI 1640 + 10% FBS[3] |
| Ca²⁺/Mg²⁺-free PBS / DPBS | Wash + resuspend 100 μL/mouse | Inoculum serum-free, no Matrigel[1] |
| Trypsin-EDTA (0.05–0.25%) | ~3–5 mL per T75 | Must be single cells; clumps acutely embolize lung |
| T75 flask or 10-cm dish | See expansion table (three dose columns) | Harvest at 85–90% confluence |
| 1 mL tuberculin syringe + 26G ½″ needle | 1 set per mouse + spares | Yang: 26G; clear bubbles[2]. Dead space can be large—prepare ~50% extra |
| Heat lamp or warm water + restrainer | Shared for the study | Dilate the lateral tail vein; Ring: ~2 min under a lamp then restrain[4]. Do not scald the tail |
| D-luciferin (Luc lines) | Per imaging SOP | Minn: 1.5 mg (15 mg/mL) retro-orbital, image at 2–5 min[1]. Dose from the protocol/insert—not invented here |
Expansion cases: mice vs T75 (three i.v. doses)
Assumes ~5×10⁶ harvested per log-phase T75 and 50% extra for Yang’s 26G tuberculin (not the flank 25G 20%). Columns = Minn 2×10⁵, this SOP typical 1×10⁶, Zhou ceiling 1.5×10⁶ (not default—mice can be in respiratory distress by week 7). A 10-cm dish is close in area.[1][2][3]
| Mice | 2×10⁵ T75 | SOP 1×10⁶ T75 | Ceiling 1.5×10⁶ T75 |
|---|---|---|---|
| 1 | 1 | 1 | 1 |
| 2 | 1 | 1 | 1 |
| 3 | 1 | 1 | 2 |
| 4 | 1 | 2 | 2 |
| 5 | 1 | 2 | 3 |
| 6 | 1 | 2 | 3 |
| 7 | 1 | 3 | 4 |
| 8 | 1 | 3 | 4 |
| 9 | 1 | 3 | 5 |
| 10 | 1 | 3 | 5 |
Inoculum recipe (per mouse + batch; no matrix)
This SOP typically uses ~2×10⁵–1×10⁶ in 100 μL PBS, no Matrigel. LM2 can colonize from 2×10³; parental 2×10⁵ often leaves an indolent residue[1]. Zhou used 1×10⁶ parental for imageable lesions; 1.5×10⁶ is a welfare ceiling. Inject promptly after mixing.[1][3][4]
| Item | This SOP (typical) | Literature variant / ceiling |
|---|---|---|
| Cells / mouse | Parental ~1×10⁶; LM2 can go to 2×10⁵ or lower | Minn parental/selection: 2×10⁵; LM2 down to 2×10³[1]. Zhou parental 1×10⁶ for lesions, 1.5×10⁶ too heavy[3]. Ring NSG 1×10⁶[4] |
| Volume | 100 μL (0.1 mL) | Minn 0.1 mL PBS[1]; Yang 100 μL[2]. Do not default to 200 μL unless the protocol says so |
| Vehicle | Serum-free PBS; no Matrigel | Minn: wash and resuspend in PBS[1]. Ring used 100 μL medium[4]. Matrix will clog lung |
| Batch multiplier | n × 1.5 (tuberculin dead space)[2] | Load one syringe per mouse so cells do not settle |
| Batch for 10 (SOP 1×10⁶) | ~1.5×10⁷ (1×10⁶ × 10 × 1.5) → T75 1×10⁶ column | 2×10⁵ arm: ~3.0×10⁶ → 1 T75 is enough |
| Viability / singles | Trypan blue ≥90%; no clumps on the scope | If <90% or clumped: do not inject; reculture |
Injection checklist (lateral tail vein, not flank or fat pad)
Warm, then inject the lateral tail vein. No anesthetic doses on this page. Do not proceed without IACUC. A white s.c. bleb = extravasation failure; acute respiratory distress is a humane endpoint.[2][4]
| Check | Pass | If fail |
|---|---|---|
| Dilation | Heat lamp or warm water until a lateral vein is obvious; Yang: dilation makes or breaks the inject[2] | Vein not visible: re-warm, switch sides—do not blindly restick the same spot |
| Restraint | Restrainer with the tail free; Ring: ~2 min under a lamp then restrain[4] | If struggling hard: stop—avoid a bent needle and air |
| Needle / air | 1 mL + 26G ½″; bevel toward the barrel marks; no bubbles[2] | Any air: re-purge. Air emboli can kill |
| Inject | Slow 100 μL into a lateral vein; vein blanches, no s.c. white bleb | White bleb / high resistance: extravasation—log as failed; do not dump the rest into tissue |
| Immediate watch | Steady breathing and activity after caging | Gasping, cyanosis, collapse → humane endpoint / vet SOP (embolus or overload) |
| Day-0 imaging (Luc) | Minn: BLI immediately after inject to drop failed xenografts[1] | No thoracic signal: exclude from the colonization arm |
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.
Follow the MC-h133 datasheet. Confluence schematic is not a real micrograph. I.v. work needs log-phase single cells. Minn used high-glucose DMEM[1].
| Item | Practice |
|---|---|
| Medium | Datasheet first. Minn: high-glucose DMEM + 10% FBS[1]. Zhou: RPMI 1640 + 10% FBS[3]. Ring: DMEM + 10% FBS[4]. ATCC classic also uses L-15 (watch CO₂). |
| 85–90% confluence (split / harvest) | Cells occupy ~85–90% of the field: nearly full, small gaps, no stacking. Overconfluence drops viability and embolizes more readily i.v. Minn counted viable cells[1]. |
| Feed / split | Medium ≥2×/week; split at 85–90%—keep log phase. After FACS enrichment of Luc/GFP lines, re-validate the metastatic phenotype[1]. |
| Passage / subline | Log parental and LM2 passages separately; do not mix doses. LM2 is an in-vivo-selected lung-tropic population[1]. |
| Day −1 | Feed; check morphology; expand enough T75s for n (see expansion table). |
What success looks like (expected, not a guarantee)
Parental vs LM2 and nude vs NSG differ by an order of magnitude—pilot first. If off-track, check extravasation/clumps before raising toward 1.5×10⁶.[1][3]
| Time point | Typical appearance |
|---|---|
| Day 0 | No s.c. bleb; steady breathing. Luc: immediate thoracic BLI[1] |
| ~Week 4 (parental 1×10⁶, nude) | Zhou SPECT often still negative—do not call “no mets” yet[3] |
| ~Weeks 6–8 (parental 1×10⁶, nude) | Lesions from week 6; many bilateral foci by week 8; the 2×10⁵ arm can be SPECT-negative with HE micromets[3] |
| LM2 (nude) | 2×10⁵ rapidly replaces parenchyma; 2×10³ can still yield aggressive lesions; parental 2×10⁵ often stays indolent. Endpoint up to 15 weeks[1] |
| NSG, 1×10⁶ (Ring control) | Large lung burden by ~5 weeks; controls euthanized ~day 57[4]. Watch endpoints earlier on NSG |
Troubleshooting
Map to flowchart nodes. Respiratory distress follows the IACUC SOP immediately—do not raise cell number to force an endpoint.
| Sign | Likely cause | Action |
|---|---|---|
| Immediate gasp / death | Clump or air embolus, volume too large, dead cells | Stop the cohort; inspect the mix; singles, no air, stay at 2×10⁵–1×10⁶ |
| White s.c. bleb on the tail | Missed the vein | Log as failed; switch sides; slow the push |
| No thoracic BLI on day 0 | Extravasation or too few cells | Exclude from colonization; audit the inject log[1] |
| Imaging-negative at week 8 | Low dose, indolent parental, or micromets only | Do HE first; Zhou 2×10⁵ is SPECT-negative with micromets[3]. QC before switching to LM2 |
| Weight drop, distress, cyanosis | Lung burden too high (esp. 1.5×10⁶ or NSG) | Humane endpoint now. Zhou 1.5×10⁶ mice were already weak by week 7[3] |
Monitoring log fields (print / copy)
No caliper volumes on this page. Luc: Minn images immediately post-inject then as scheduled; Ring weekly for 5 weeks[1][4]. Breathing and weight outrank the image.
| Field | How to log |
|---|---|
| Date / day post-inject | D0 = tail-vein day |
| Mouse / cage / arm / subline | Split parental vs LM2; lock IDs after randomization |
| Vein success | Yes / extravasation fail; Luc: log D0 thoracic signal |
| Body weight (g) | Same day as imaging; sharp drop triggers endpoint |
| Breathing / activity | Normal / gasp / cyanosis; stop and follow SOP if abnormal |
| BLI (Luc) | Thoracic ROI; same pose. Substrate per imaging SOP |
Literature case comparison
This SOP typically uses parental 1×10⁶ / 100 μL PBS; LM2 uses a lower dose. The T75 table stocks to 1.5×10⁶ but that dose is not the default.
| Paper | Host | Cells | Volume / needle / vehicle | Readout |
|---|---|---|---|---|
| Minn 2005[1] | ♀ Balb/c nude, 4–6 wk | 2×10⁵; LM2 down to 2×10³ | 0.1 mL PBS; lateral tail vein | BLI + gross lung; endpoint 15 wk |
| Yang 2012[2] | ♀ NOD-SCID, 4–6 wk | Luc-MDA-MB-231 (volume/needle in the chapter; cell number not in the abstract) | 100 μL; 1 mL + 26G ½″; heat dilation | Noninvasive BLI of lung colonization |
| Zhou 2012[3] | ♀ nude, 4–5 wk | 2×10⁵ / 1×10⁶ / 1.5×10⁶ | Tail vein (volume not stated; this SOP aligns to 100 μL) | SPECT/CT + HE; 1×10⁶ lesions by wk 8; 1.5×10⁶ weak by wk 7 |
| Ring 2020[4] | ♀ NSG, 8–10 wk | 1×10⁶ | 100 μL medium; heat lamp 2 min + restrainer | Weekly BLI 5 wk; controls euthanized ~day 57 |
- 01[Ethics/facility] Obtain IACUC approval; quarantine female immunodeficient mice ≥3–7 days under SPF. Write humane endpoints for respiratory distress, cyanosis, and sharp weight loss. See unpack list and host table. RUO. This page does not replace tail-vein training or the veterinary SOP.
SKUs link to catalog items. This route is a single-cell i.v. suspension—no Matrigel (pulmonary emboli). Follow the MC-h133 datasheet.[1][2][3]
Item Per mouse / study Notes MDA-MB-231 or LM2 subline MC-h133 Start from 1 vial STR / mycoplasma-qualified; log passage; LM2 colonizes lung far better than parental[1] FBS (routine culture) MC100 10% in complete medium Prefer regular grade Medium (DMEM or RPMI) ~15 mL per T75 Minn: high-glucose DMEM + 10% FBS[1]; Zhou: RPMI 1640 + 10% FBS[3] Ca²⁺/Mg²⁺-free PBS / DPBS Wash + resuspend 100 μL/mouse Inoculum serum-free, no Matrigel[1] Trypsin-EDTA (0.05–0.25%) ~3–5 mL per T75 Must be single cells; clumps acutely embolize lung T75 flask or 10-cm dish See expansion table (three dose columns) Harvest at 85–90% confluence 1 mL tuberculin syringe + 26G ½″ needle 1 set per mouse + spares Yang: 26G; clear bubbles[2]. Dead space can be large—prepare ~50% extra Heat lamp or warm water + restrainer Shared for the study Dilate the lateral tail vein; Ring: ~2 min under a lamp then restrain[4]. Do not scald the tail D-luciferin (Luc lines) Per imaging SOP Minn: 1.5 mg (15 mg/mL) retro-orbital, image at 2–5 min[1]. Dose from the protocol/insert—not invented here Warm, then inject the lateral tail vein. No anesthetic doses on this page. Do not proceed without IACUC. A white s.c. bleb = extravasation failure; acute respiratory distress is a humane endpoint.[2][4]
Check Pass If fail Dilation Heat lamp or warm water until a lateral vein is obvious; Yang: dilation makes or breaks the inject[2] Vein not visible: re-warm, switch sides—do not blindly restick the same spot Restraint Restrainer with the tail free; Ring: ~2 min under a lamp then restrain[4] If struggling hard: stop—avoid a bent needle and air Needle / air 1 mL + 26G ½″; bevel toward the barrel marks; no bubbles[2] Any air: re-purge. Air emboli can kill Inject Slow 100 μL into a lateral vein; vein blanches, no s.c. white bleb White bleb / high resistance: extravasation—log as failed; do not dump the rest into tissue Immediate watch Steady breathing and activity after caging Gasping, cyanosis, collapse → humane endpoint / vet SOP (embolus or overload) Day-0 imaging (Luc) Minn: BLI immediately after inject to drop failed xenografts[1] No thoracic signal: exclude from the colonization arm Map to flowchart nodes. Respiratory distress follows the IACUC SOP immediately—do not raise cell number to force an endpoint.
Sign Likely cause Action Immediate gasp / death Clump or air embolus, volume too large, dead cells Stop the cohort; inspect the mix; singles, no air, stay at 2×10⁵–1×10⁶ White s.c. bleb on the tail Missed the vein Log as failed; switch sides; slow the push No thoracic BLI on day 0 Extravasation or too few cells Exclude from colonization; audit the inject log[1] Imaging-negative at week 8 Low dose, indolent parental, or micromets only Do HE first; Zhou 2×10⁵ is SPECT-negative with micromets[3]. QC before switching to LM2 Weight drop, distress, cyanosis Lung burden too high (esp. 1.5×10⁶ or NSG) Humane endpoint now. Zhou 1.5×10⁶ mice were already weak by week 7[3] - 02[Cells / Expand MDA-MB-231 ± Luc/LM2] STR/mycoplasma-qualified MC-h133; log parental and LM2 passages separately. Feed on D−1; harvest at 85–90% confluence. Validate that Luc/GFP does not change metastatic phenotype[1]. See culture card and T75 table (2×10⁵ / 1×10⁶ / 1.5×10⁶—do not reuse flank flask math).[1][2][3]
Assumes ~5×10⁶ harvested per log-phase T75 and 50% extra for Yang’s 26G tuberculin (not the flank 25G 20%). Columns = Minn 2×10⁵, this SOP typical 1×10⁶, Zhou ceiling 1.5×10⁶ (not default—mice can be in respiratory distress by week 7). A 10-cm dish is close in area.[1][2][3]
Mice 2×10⁵ T75 SOP 1×10⁶ T75 Ceiling 1.5×10⁶ T75 1 1 1 1 2 1 1 1 3 1 1 2 4 1 2 2 5 1 2 3 6 1 2 3 7 1 3 4 8 1 3 4 9 1 3 5 10 1 3 5 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.
Click to enlargeLarge gaps; wait one more day Nearly full, small gaps; no stacking No gaps; do not inject Follow the MC-h133 datasheet. Confluence schematic is not a real micrograph. I.v. work needs log-phase single cells. Minn used high-glucose DMEM[1].
Item Practice Medium Datasheet first. Minn: high-glucose DMEM + 10% FBS[1]. Zhou: RPMI 1640 + 10% FBS[3]. Ring: DMEM + 10% FBS[4]. ATCC classic also uses L-15 (watch CO₂). 85–90% confluence (split / harvest) Cells occupy ~85–90% of the field: nearly full, small gaps, no stacking. Overconfluence drops viability and embolizes more readily i.v. Minn counted viable cells[1]. Feed / split Medium ≥2×/week; split at 85–90%—keep log phase. After FACS enrichment of Luc/GFP lines, re-validate the metastatic phenotype[1]. Passage / subline Log parental and LM2 passages separately; do not mix doses. LM2 is an in-vivo-selected lung-tropic population[1]. Day −1 Feed; check morphology; expand enough T75s for n (see expansion table). - 03[Single-cell suspension] PBS wash, trypsinize to single cells, neutralize, centrifuge; resuspend in serum-free PBS. Trypan blue ≥90%, no clumps. No Matrigel. Ice; inject promptly. Batch math on the recipe card (~50% extra).
This SOP typically uses ~2×10⁵–1×10⁶ in 100 μL PBS, no Matrigel. LM2 can colonize from 2×10³; parental 2×10⁵ often leaves an indolent residue[1]. Zhou used 1×10⁶ parental for imageable lesions; 1.5×10⁶ is a welfare ceiling. Inject promptly after mixing.[1][3][4]
Item This SOP (typical) Literature variant / ceiling Cells / mouse Parental ~1×10⁶; LM2 can go to 2×10⁵ or lower Minn parental/selection: 2×10⁵; LM2 down to 2×10³[1]. Zhou parental 1×10⁶ for lesions, 1.5×10⁶ too heavy[3]. Ring NSG 1×10⁶[4] Volume 100 μL (0.1 mL) Minn 0.1 mL PBS[1]; Yang 100 μL[2]. Do not default to 200 μL unless the protocol says so Vehicle Serum-free PBS; no Matrigel Minn: wash and resuspend in PBS[1]. Ring used 100 μL medium[4]. Matrix will clog lung Batch multiplier n × 1.5 (tuberculin dead space)[2] Load one syringe per mouse so cells do not settle Batch for 10 (SOP 1×10⁶) ~1.5×10⁷ (1×10⁶ × 10 × 1.5) → T75 1×10⁶ column 2×10⁵ arm: ~3.0×10⁶ → 1 T75 is enough Viability / singles Trypan blue ≥90%; no clumps on the scope If <90% or clumped: do not inject; reculture - 04
- 05[Tail-vein inject] Follow the injection checklist: warm, restrain, 26G, 100 μL, no bubbles, no s.c. bleb[2][4]. Anesthetic doses come only from the IACUC SOP.
SKUs link to catalog items. This route is a single-cell i.v. suspension—no Matrigel (pulmonary emboli). Follow the MC-h133 datasheet.[1][2][3]
Item Per mouse / study Notes MDA-MB-231 or LM2 subline MC-h133 Start from 1 vial STR / mycoplasma-qualified; log passage; LM2 colonizes lung far better than parental[1] FBS (routine culture) MC100 10% in complete medium Prefer regular grade Medium (DMEM or RPMI) ~15 mL per T75 Minn: high-glucose DMEM + 10% FBS[1]; Zhou: RPMI 1640 + 10% FBS[3] Ca²⁺/Mg²⁺-free PBS / DPBS Wash + resuspend 100 μL/mouse Inoculum serum-free, no Matrigel[1] Trypsin-EDTA (0.05–0.25%) ~3–5 mL per T75 Must be single cells; clumps acutely embolize lung T75 flask or 10-cm dish See expansion table (three dose columns) Harvest at 85–90% confluence 1 mL tuberculin syringe + 26G ½″ needle 1 set per mouse + spares Yang: 26G; clear bubbles[2]. Dead space can be large—prepare ~50% extra Heat lamp or warm water + restrainer Shared for the study Dilate the lateral tail vein; Ring: ~2 min under a lamp then restrain[4]. Do not scald the tail D-luciferin (Luc lines) Per imaging SOP Minn: 1.5 mg (15 mg/mL) retro-orbital, image at 2–5 min[1]. Dose from the protocol/insert—not invented here Warm, then inject the lateral tail vein. No anesthetic doses on this page. Do not proceed without IACUC. A white s.c. bleb = extravasation failure; acute respiratory distress is a humane endpoint.[2][4]
Check Pass If fail Dilation Heat lamp or warm water until a lateral vein is obvious; Yang: dilation makes or breaks the inject[2] Vein not visible: re-warm, switch sides—do not blindly restick the same spot Restraint Restrainer with the tail free; Ring: ~2 min under a lamp then restrain[4] If struggling hard: stop—avoid a bent needle and air Needle / air 1 mL + 26G ½″; bevel toward the barrel marks; no bubbles[2] Any air: re-purge. Air emboli can kill Inject Slow 100 μL into a lateral vein; vein blanches, no s.c. white bleb White bleb / high resistance: extravasation—log as failed; do not dump the rest into tissue Immediate watch Steady breathing and activity after caging Gasping, cyanosis, collapse → humane endpoint / vet SOP (embolus or overload) Day-0 imaging (Luc) Minn: BLI immediately after inject to drop failed xenografts[1] No thoracic signal: exclude from the colonization arm - 06[Monitor] Weight and breathing first. Luc: Minn BLI immediately post-inject to drop failed xenografts[1], then as scheduled. Fields on the monitor template.
No caliper volumes on this page. Luc: Minn images immediately post-inject then as scheduled; Ring weekly for 5 weeks[1][4]. Breathing and weight outrank the image.
Field How to log Date / day post-inject D0 = tail-vein day Mouse / cage / arm / subline Split parental vs LM2; lock IDs after randomization Vein success Yes / extravasation fail; Luc: log D0 thoracic signal Body weight (g) Same day as imaging; sharp drop triggers endpoint Breathing / activity Normal / gasp / cyanosis; stop and follow SOP if abnormal BLI (Luc) Thoracic ROI; same pose. Substrate per imaging SOP Map to flowchart nodes. Respiratory distress follows the IACUC SOP immediately—do not raise cell number to force an endpoint.
Sign Likely cause Action Immediate gasp / death Clump or air embolus, volume too large, dead cells Stop the cohort; inspect the mix; singles, no air, stay at 2×10⁵–1×10⁶ White s.c. bleb on the tail Missed the vein Log as failed; switch sides; slow the push No thoracic BLI on day 0 Extravasation or too few cells Exclude from colonization; audit the inject log[1] Imaging-negative at week 8 Low dose, indolent parental, or micromets only Do HE first; Zhou 2×10⁵ is SPECT-negative with micromets[3]. QC before switching to LM2 Weight drop, distress, cyanosis Lung burden too high (esp. 1.5×10⁶ or NSG) Humane endpoint now. Zhou 1.5×10⁶ mice were already weak by week 7[3] - 07[Endpoint] Gross lung + HE after euthanasia; still do histology if imaging is negative (micromets)[3]. This model omits intravasation—use the spontaneous orthotopic-mets protocol for the full cascade.
- 08[QC] Fix strain, sex, age, subline, passage, and cell number; independent repeats. Troubleshoot before changing dose.
No caliper volumes on this page. Luc: Minn images immediately post-inject then as scheduled; Ring weekly for 5 weeks[1][4]. Breathing and weight outrank the image.
Field How to log Date / day post-inject D0 = tail-vein day Mouse / cage / arm / subline Split parental vs LM2; lock IDs after randomization Vein success Yes / extravasation fail; Luc: log D0 thoracic signal Body weight (g) Same day as imaging; sharp drop triggers endpoint Breathing / activity Normal / gasp / cyanosis; stop and follow SOP if abnormal BLI (Luc) Thoracic ROI; same pose. Substrate per imaging SOP
Reagents / materials
| Item | Role | Conc. / dose |
|---|---|---|
| MDA-MB-231 cells (MC-h133) | Tail-vein inoculum (parental or LM2) | 本 SOP 亲本约 1×10⁶ / 100 μL PBS；LM2 更低；上限 1.5×10⁶ 不作默认[[3]] |
| Fetal bovine serum (FBS) | 10% complete medium | 常规培养优先 MC100 |
| Luc/GFP line (optional) | In vivo imaging | 验证不影响转移表型[[1]] |
| D-luciferin (for Luc) | BLI substrate | Minn：1.5 mg（15 mg/mL）眶后[[1]]；其余按成像 SOP |
| Test drug / vehicle (optional) | Treatment arm | 按药理方案 |
Readouts
Lung colony number/area, BLI signal, weight, survival, lung pathology.
References (PubMed)
- [1]PMID 16049480 — Genes that mediate breast cancer metastasis to lung. Nature (2005)
- [2]PMID 22956145 — Mouse models for tumor metastasis. Methods Mol Biol (2012)
- [3]PMID 22737193 — Monitoring Breast Tumor Lung Metastasis by U-SPECT-II/CT with an Integrin α(v)β(3)-Targeted Radiotracer( 99m)Tc-3P-RGD(2). Theranostics (2012)
- [4]PMID 33167919 — EP300 knockdown reduces cancer stem cell phenotype, tumor growth and metastasis in triple negative breast cancer. BMC Cancer (2020)
Disclaimer: RUO; IACUC required. Optimize by strain and pilot. Red tags mark weak or non-metastatic parental endpoints.