Use cases
Orthotopic engraftment of human TNBC line MDA-MB-231 (MC-h133) into the #4 mammary fat pad of immunodeficient mice. Written as a novice pack (BOM, timeline, recipe, surgery checks) from the literature—not a substitute for IACUC or surgical training.
Catalog: MC-h133 · Product modeling page · In-vitro spheroid
Research use only (RUO)—not clinical care. IACUC approval, SPF housing, and surgical training are required. This page does not give anesthetic/analgesic doses and does not replace the veterinary SOP or JoVE videos. Do not proceed without approval.
Kocatürk JoVE: fat-pad injection (video) · PMID 25742185Cheng JoVE: MDA-MB-231 orthotopic TNBC (T75 + dead-space note) · PMID 32250353
Use-case overview
Use this page for local orthotopic growth; switch protocols for lung/bone/brain mets—do not reuse this SOP as-is.
| Use | Fit | Primary readout | Notes |
|---|---|---|---|
| Orthotopic local growth / TNBC efficacy | Recommended | Primary volume/weight, pathology[1][3] | #4 fat pad; female nude or NSG. |
| Spontaneous mets after orthotopic growth | Suitable | Long-term lung/node follow-up[1][4] | Same inoculum skeleton, longer follow-up; see the spontaneous-mets protocol. |
| Tail-vein lung / intracardiac bone / brain mets | Not recommended | Colonization, not the orthotopic niche | Use the matching route protocol—not this surgery SOP. |
Does the workflow match?
Efficacy and QC share this inoculum skeleton; metastasis endpoints need another protocol.
| Use | vs foolproof SOP | Differences vs core SOP |
|---|---|---|
| Orthotopic growth / efficacy | Same core SOP | This page is the core SOP. |
| Spontaneous mets after orthotopic growth | Core + add-ons | Same inject; longer follow-up or primary resection. |
| Experimental lung / bone / brain mets | Other route | Tail vein, intracardiac, or intracranial—not fat pad. |
Host spec: sex × strain × age
Kocatürk used NSG[2], Price used nude mice[1]; this SOP defaults to female nudes. Do not mix cell numbers across hosts. Quarantine per facility SOP, often ≥3–7 days. RUO.
| Indication / context | Sex | Strain / host | Notes |
|---|---|---|---|
| This SOP default: orthotopic growth / efficacy | Female (required) | Nude nu/nu; 6–8 weeks; quarantine ≥3–7 days | #4 fat pad; ~1×10⁶ cells; pilot n=3–5, efficacy often 6–8/arm (stats + IACUC)[1]. |
| Higher take / JoVE orthotopic demo | Female | NSG (NOD.Cg-Prkdc scid Il2rg) | Kocatürk: 5×10⁵ in ≤150 μL ± Matrigel[2]. Deeper immunodeficiency often steadies take. |
| BLI / Luc line | Female | Nude, NOD-SCID, or NSG | Confirm the label does not alter growth/mets, or run parental controls[4]. |
| Male | Not recommended | — | Fat-pad models are female-standard; males need written justification and do not extrapolate cleanly. |
In one line
IACUC → expand qualified cells → mammary fat-pad inject → caliper/weight → optional therapy → endpoint pathology.
D−1 / D0 timeline
1. D−7 to D−3
SPF quarantine; confirm female/age; IACUC in hand.
2. D−1
Feed cells at 85–90% confluence; clip #4 nipple to midline; weigh[[2]].
3. D0 morning
PBS wash → trypsin → neutralize/spin → serum-free resuspend → count (≥90% viable).
4. D0 mix
Mix per recipe + Matrigel card (+ 40–50% extra); stay on ice. Instruments sterile.
5. D0 inject
Anesthetize → #4 pad → slow inject to bleb → close. Finish within 30–60 min.
6. D0 recovery
Warm until righting; analgesia per SOP; log dose and passage.
7. From D+3
Caliper + weight 2–3×/week; stop at humane endpoints.
Protocol overview
Scenario-specific flowchart (core engraftment skeleton with host/therapy or imaging/readout changes).
- IACUC + SPF nude quarantine
- Expand MDA-MB-231 (log passage; conservative T75: 1 mouse → 1 flask, 10 → 6)
- Day-of count (viability ≥90%)
- Anesthetize; expose #4 fat pad
- Inject ~1×10⁶ cells (± Matrigel)
- Close incision; warm recovery
- Weekly caliper + body weight
- Optional 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; others are lab-standard. Follow the MC-h133 datasheet for medium; JoVE often uses RPMI/DMEM + 10% FBS.[2][3]
| Item | Per mouse / study | Notes |
|---|---|---|
| MDA-MB-231 cells MC-h133 | Start from 1 vial | STR / mycoplasma-qualified; log passage |
| FBS (routine culture) MC100 | 10% in complete medium | Prefer regular grade; HI only if datasheet says so |
| Medium (L-15 / DMEM / RPMI) | ~15 mL per T75 | ATCC classic is L-15; JoVE often RPMI or DMEM + CO₂ |
| Ca²⁺/Mg²⁺-free PBS / DPBS | Wash + resuspend; inject volume on recipe card | Keep inoculum serum-free |
| Trypsin-EDTA (0.05–0.25%) | ~3–5 mL per T75 | Single cells; avoid over-trypsinizing |
| Matrigel / BMM (optional) | 25–50 μL/mouse (if 1:1 in 50–100 μL total) | On ice; step-by-step on the Matrigel card[2][3] |
| T75 flask or 10-cm dish | See expansion table (1 mouse ≥1 flask; 10 ≥6) | Harvest at 85–90% confluence |
| Tuberculin syringe 26–27G | 1 per mouse + spares | Dead space can be ~70 μL; prepare 40–50% extra mix[3] |
| Suture or tissue glue, sterile instruments | Per surgical n | Autoclave; clip #4 nipple area |
| Caliper | Shared for the study | Volume ≈ L × W² / 2 |
Expansion cases: nude mice vs T75 flasks
Inoculum recipe (per mouse + batch)
This SOP defaults to ~1×10⁶ in 50–100 μL. Conservative flask counts use 2×10⁶/mouse + 40–50% dead space (same as the T75 table). Keep on ice; inject within 30–60 min.[1][2][3]
| Item | This SOP (typical) | Most conservative (JoVE high dose) |
|---|---|---|
| Cells / mouse | ~1×10⁶ | 2×10⁶[3] (Kocatürk used 5×10⁵[2]) |
| Volume / mouse | 50–100 μL | 100 μL (0.1 mL)[3]; ≤150 μL[2] |
| Matrix | Optional 1:1 Matrigel (see Matrigel card) | PBS + 25% BMM[3], or optional Matrigel[2] |
| Batch multiplier | n × 1.0 | n × 1.4–1.5 (dead space + loss)[3] |
| Batch for 10 mice | ~1.0×10⁷ (no extra) | ~3.0×10⁷ (2×10⁶ × 10 × 1.5) → ≥6 T75 |
| Viability | Trypan blue ≥90% | Do not inject if <90%; reculture |
Matrigel / BMM: how to use it in this experiment
Matrix is optional for this orthotopic SOP. Cheng JoVE resuspends MDA-MB-231 in ice-cold PBS + 25% basement-membrane matrix; Kocatürk lists PBS, medium, or Matrigel as options. Cold handling follows the product insert; ratios and needle dwell are from the papers, not a substitute for the manufacturer sheet.[2][3]
| Item | Practice in this experiment |
|---|---|
| Use it or skip it | Use it to reduce leak and steady take: it polymerizes in the pad[2]. This SOP’s common option is 1:1; to match Cheng, use 25% matrix in 100 μL with 2×10⁶ cells[3]. One choice for the whole study; log the lot. |
| When to skip it | If the question is integrin / ECM / stroma crosstalk, use PBS or medium[2]. Standard lots contain growth factors that can bias pharmacology; prefer GFR (Kocatürk lists Corning 356230)[2]. |
| Thaw and store | Follow the insert: typically −20 °C stock; thaw fully at 4 °C / on ice (often overnight)—do not thaw at room temperature or in a water bath until it gels. Aliquot on ice to limit freeze–thaw. Keep on ice while in use[3]. |
| Chill the hardware | Pre-chill tips, tubes, syringes/needles, PBS, and the cell pellet. Matrix thickens as soon as it leaves ice; finish warm steps (trypsin, neutralize, count) before mixing. |
| Mix A (Cheng) | After counting, adjust to 20×10⁶ / mL in ice-cold 1× PBS containing 25% basement-membrane matrix. Inject 100 μL = 2×10⁶ cells per mouse (~75 μL cell-PBS + 25 μL matrix). Keep the mix on ice[3]. |
| Mix B (this SOP, 1:1) | After counting, resuspend at 2× final density in ice-cold serum-free PBS, then gently mix 1:1 with ice-cold Matrigel (no foam). Final 50–100 μL/mouse, ~1×10⁶ cells. Kocatürk says optional Matrigel resuspension without a fixed ratio[2]. |
| Worked example (mix A, 10 mice + dead space) | 10 × 2×10⁶ × 1.5 = 3.0×10⁷ cells in 1.5 mL (10 × 100 μL × 1.5). Matrix = 1.5 mL × 25% = 375 μL; the rest is ice-cold PBS + cells. The extra 40–50% covers tuberculin dead space (up to ~70 μL) and loss[3]. |
| Load and inject | Draw with a pre-chilled syringe; clear bubbles. Keep an ice box at the table. After the bleb, wait 10–15 s before withdrawing[3]. Finish in 30–60 min. If the mix strings, thickens, or clogs, discard the tube—do not dilute set gel. |
Surgery checklist (#4 mammary fat pad)
Watch the JoVE video first. No anesthetic doses on this page. Do not proceed without IACUC and surgical training. Leak or no bleb = failed inoculum.[2][3]
| Check | Pass | If fail |
|---|---|---|
| Landmark | Female #4 nipple (caudal pair); clip to midline[2] | Stop if the landmark is wrong—do not inject blindly |
| Approach | Small incision to expose the pad, or slow percutaneous inject (SOP / JoVE) | Large incision or ongoing bleed → hemostasis, then decide |
| Needle | 26–27G tuberculin; clear air bubbles (Cheng video also uses 25G)[3] | Clog: ice the mix—Matrigel may have set |
| Needle dwell if matrix used | Wait 10–15 s after the push for the matrix to stiffen, then withdraw to limit leak[3] | Immediate withdraw + leak: log as failed inject |
| Inject | Slow push until a slight bleb; avoid muscle and peritoneum | Leak / no bleb: exclude or reinject only if the protocol allows |
| Close / recover | Suture or glue; warm until righting reflex; analgesia per IACUC | Prolonged recovery or respiratory depression → humane endpoint / 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.
Follow the MC-h133 datasheet. Confluence = % of growth area covered by attached cells; if the monolayer is even, that is about the fraction of the inverted-microscope field occupied by cells. Schematic, not a real micrograph.[3]
| Item | Practice |
|---|---|
| Medium | Datasheet first. JoVE: RPMI 1640 + 10% FBS, 37°C, 5% CO₂, ~15 mL in T75[3]. ATCC classic also uses L-15 + 10% FBS (watch CO₂). |
| 85–90% confluence (split / harvest) | Under the inverted microscope cells occupy ~85–90% of the field: nearly full, with small gaps, no stacking or peeling. MDA-MB-231 is spindle-like, so 90% looks looser than cobblestone epithelia—judge by gaps, do not wait for 100%. ~50% is too sparse for yield; a packed monolayer drops viability and should not be injected.[3] |
| Feed / split | Medium ≥2×/week; split at 85–90% confluence as above—keep log phase, do not overgrow[3]. |
| Passage | Fix an early–mid window for in-vivo work and log it; validate labeled lines vs parental[4]. |
| Day −1 | Feed; check morphology; expand enough T75s for n (see expansion table). |
What success looks like (expected, not a guarantee)
Kinetics vary by strain, passage, and dose—pilot first. If off-track, use the troubleshooting table before raising cell number.[1][3]
| Time point | Typical appearance |
|---|---|
| Day 0 | Local bleb; no ongoing leak; mouse active after righting |
| ~Weeks 1–2 | Palpable orthotopic nodule more common at 1–2×10⁶; log first measurable day |
| ~Weeks 2–4 | Volume curve in hand; efficacy often randomizes at 100–150 mm³ (per protocol) |
| Take rate | Parental MDA-MB-231 takes well orthotopically in nude/NSG[1], but lots/passage vary; pilot n ≈ 3–5 |
Troubleshooting
Map to flowchart nodes. Welfare issues follow the IACUC SOP—do not improvise doses from this page.
| Sign | Likely cause | Action |
|---|---|---|
| Clumps / clogged needle | Incomplete digest; Matrigel warmed | Gentle triturate or filter; ice the tube; discard set matrix |
| Viability <90% | Overconfluence, over-trypsin, too long off ice | Do not inject; reculture in log phase and recount |
| Leak / no bleb | Volume too large, too fast, wrong plane | Log as failed inject; reduce volume and slow the push |
| No palpable tumor by week 2 | Missed inject, too few cells, late passage, strain mismatch | Audit records; QC before raising dose toward the literature ceiling |
| Ulcer, sharp weight loss, distress | Humane endpoint | Euthanize per protocol immediately—do not delay for a time point |
Monitoring log fields (print / copy)
Measure 2–3×/week. Volume ≈ L × W² / 2. Enrollment volume and dosing route live in the IACUC protocol—not invented here.
| Field | How to log |
|---|---|
| Date / day post-inject | D0 = inoculation day |
| Mouse / cage / arm | Lock IDs after randomization |
| L, W (mm) | Same operator and caliper; longest axis |
| Volume (mm³) | L × W² / 2 |
| Body weight (g) | Same day as caliper |
| Ulcer / activity / breathing | Y/N + brief note; stop if endpoint hit |
Literature case comparison
This SOP uses a mid dose; the T75 table stocks to the conservative (right-hand) column. Open PMIDs for the papers / JoVE videos.
| Paper | Host | Cells | Volume / matrix | Readout |
|---|---|---|---|---|
| Price 1990[1] | Nude mammary fat pad | Often ~1×10⁶ class | Orthotopic | Tumorigenicity / mets spectrum |
| Kocatürk 2015 JoVE[2] | NSG; method fits other immunodeficient hosts | 5×10⁵ / mouse | ≤150 μL; optional Matrigel | Orthotopic growth; optional mets harvest |
| Cheng 2020 JoVE[3] | #4 fat pad | 2×10⁶ / mouse | 100 μL PBS + 25% BMM; T75 expansion | Caliper + lung imaging; prepare 40–50% extra mix |
| Jenkins 2005[4] | Immunodeficient | Luc-labeled line | BLI | Confirm the label does not change phenotype |
- 01[Ethics/facility] Obtain IACUC approval; quarantine female nude (nu/nu) mice ≥3–7 days under SPF. See unpack list and host table. Handle MDA-MB-231 per biosafety. RUO. This page does not replace surgical training or the veterinary SOP.
SKUs link to catalog items; others are lab-standard. Follow the MC-h133 datasheet for medium; JoVE often uses RPMI/DMEM + 10% FBS.[2][3]
Item Per mouse / study Notes MDA-MB-231 cells MC-h133 Start from 1 vial STR / mycoplasma-qualified; log passage FBS (routine culture) MC100 10% in complete medium Prefer regular grade; HI only if datasheet says so Medium (L-15 / DMEM / RPMI) ~15 mL per T75 ATCC classic is L-15; JoVE often RPMI or DMEM + CO₂ Ca²⁺/Mg²⁺-free PBS / DPBS Wash + resuspend; inject volume on recipe card Keep inoculum serum-free Trypsin-EDTA (0.05–0.25%) ~3–5 mL per T75 Single cells; avoid over-trypsinizing Matrigel / BMM (optional) 25–50 μL/mouse (if 1:1 in 50–100 μL total) On ice; step-by-step on the Matrigel card[2][3] T75 flask or 10-cm dish See expansion table (1 mouse ≥1 flask; 10 ≥6) Harvest at 85–90% confluence Tuberculin syringe 26–27G 1 per mouse + spares Dead space can be ~70 μL; prepare 40–50% extra mix[3] Suture or tissue glue, sterile instruments Per surgical n Autoclave; clip #4 nipple area Caliper Shared for the study Volume ≈ L × W² / 2 - 02[Cells / Expand MDA-MB-231 (log passage)] STR/mycoplasma-qualified MC-h133; fixed passage. Feed on D−1; harvest at 85–90% confluence. See culture card and T75 table.[1][2][3]
Most conservative: 2×10⁶ cells/mouse (JoVE) + 50% syringe dead-space/loss; log-phase T75 counted at ~5×10⁶ harvested, rounded up. A 10-cm dish is close in area—plan the same count.[2][3]
Nude mice T75 flasks 1 1 2 2 3 2 4 3 5 3 6 4 7 5 8 5 9 6 10 6 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 = % of growth area covered by attached cells; if the monolayer is even, that is about the fraction of the inverted-microscope field occupied by cells. Schematic, not a real micrograph.[3]
Item Practice Medium Datasheet first. JoVE: RPMI 1640 + 10% FBS, 37°C, 5% CO₂, ~15 mL in T75[3]. ATCC classic also uses L-15 + 10% FBS (watch CO₂). 85–90% confluence (split / harvest) Under the inverted microscope cells occupy ~85–90% of the field: nearly full, with small gaps, no stacking or peeling. MDA-MB-231 is spindle-like, so 90% looks looser than cobblestone epithelia—judge by gaps, do not wait for 100%. ~50% is too sparse for yield; a packed monolayer drops viability and should not be injected.[3] Feed / split Medium ≥2×/week; split at 85–90% confluence as above—keep log phase, do not overgrow[3]. Passage Fix an early–mid window for in-vivo work and log it; validate labeled lines vs parental[4]. Day −1 Feed; check morphology; expand enough T75s for n (see expansion table). - 03[Harvest] PBS wash, trypsinize to single cells, neutralize, centrifuge; resuspend serum-free. Trypan blue ≥90%. Ice; inject within 30–60 min. Batch math on the recipe card.
- 04[Dose/volume] This SOP ~1×10⁶ in 50–100 μL (± 1:1 Matrigel). To match Cheng: 2×10⁶ in 100 μL + 25% matrix with 40–50% extra mix. Mixing, ice, and needle dwell are on the Matrigel card. Avoid bubbles.[2][3]
This SOP defaults to ~1×10⁶ in 50–100 μL. Conservative flask counts use 2×10⁶/mouse + 40–50% dead space (same as the T75 table). Keep on ice; inject within 30–60 min.[1][2][3]
Item This SOP (typical) Most conservative (JoVE high dose) Cells / mouse ~1×10⁶ 2×10⁶[3] (Kocatürk used 5×10⁵[2]) Volume / mouse 50–100 μL 100 μL (0.1 mL)[3]; ≤150 μL[2] Matrix Optional 1:1 Matrigel (see Matrigel card) PBS + 25% BMM[3], or optional Matrigel[2] Batch multiplier n × 1.0 n × 1.4–1.5 (dead space + loss)[3] Batch for 10 mice ~1.0×10⁷ (no extra) ~3.0×10⁷ (2×10⁶ × 10 × 1.5) → ≥6 T75 Viability Trypan blue ≥90% Do not inject if <90%; reculture Matrix is optional for this orthotopic SOP. Cheng JoVE resuspends MDA-MB-231 in ice-cold PBS + 25% basement-membrane matrix; Kocatürk lists PBS, medium, or Matrigel as options. Cold handling follows the product insert; ratios and needle dwell are from the papers, not a substitute for the manufacturer sheet.[2][3]
Item Practice in this experiment Use it or skip it Use it to reduce leak and steady take: it polymerizes in the pad[2]. This SOP’s common option is 1:1; to match Cheng, use 25% matrix in 100 μL with 2×10⁶ cells[3]. One choice for the whole study; log the lot. When to skip it If the question is integrin / ECM / stroma crosstalk, use PBS or medium[2]. Standard lots contain growth factors that can bias pharmacology; prefer GFR (Kocatürk lists Corning 356230)[2]. Thaw and store Follow the insert: typically −20 °C stock; thaw fully at 4 °C / on ice (often overnight)—do not thaw at room temperature or in a water bath until it gels. Aliquot on ice to limit freeze–thaw. Keep on ice while in use[3]. Chill the hardware Pre-chill tips, tubes, syringes/needles, PBS, and the cell pellet. Matrix thickens as soon as it leaves ice; finish warm steps (trypsin, neutralize, count) before mixing. Mix A (Cheng) After counting, adjust to 20×10⁶ / mL in ice-cold 1× PBS containing 25% basement-membrane matrix. Inject 100 μL = 2×10⁶ cells per mouse (~75 μL cell-PBS + 25 μL matrix). Keep the mix on ice[3]. Mix B (this SOP, 1:1) After counting, resuspend at 2× final density in ice-cold serum-free PBS, then gently mix 1:1 with ice-cold Matrigel (no foam). Final 50–100 μL/mouse, ~1×10⁶ cells. Kocatürk says optional Matrigel resuspension without a fixed ratio[2]. Worked example (mix A, 10 mice + dead space) 10 × 2×10⁶ × 1.5 = 3.0×10⁷ cells in 1.5 mL (10 × 100 μL × 1.5). Matrix = 1.5 mL × 25% = 375 μL; the rest is ice-cold PBS + cells. The extra 40–50% covers tuberculin dead space (up to ~70 μL) and loss[3]. Load and inject Draw with a pre-chilled syringe; clear bubbles. Keep an ice box at the table. After the bleb, wait 10–15 s before withdrawing[3]. Finish in 30–60 min. If the mix strings, thickens, or clogs, discard the tube—do not dilute set gel. - 05[Surgery] Follow the surgery checklist: #4 nipple, 26–27G, slow bleb, close and warm. Anesthetic/analgesic doses come only from the IACUC SOP. Watch JoVE (Price 1990; Kocatürk 2015; Cheng 2020).
SKUs link to catalog items; others are lab-standard. Follow the MC-h133 datasheet for medium; JoVE often uses RPMI/DMEM + 10% FBS.[2][3]
Item Per mouse / study Notes MDA-MB-231 cells MC-h133 Start from 1 vial STR / mycoplasma-qualified; log passage FBS (routine culture) MC100 10% in complete medium Prefer regular grade; HI only if datasheet says so Medium (L-15 / DMEM / RPMI) ~15 mL per T75 ATCC classic is L-15; JoVE often RPMI or DMEM + CO₂ Ca²⁺/Mg²⁺-free PBS / DPBS Wash + resuspend; inject volume on recipe card Keep inoculum serum-free Trypsin-EDTA (0.05–0.25%) ~3–5 mL per T75 Single cells; avoid over-trypsinizing Matrigel / BMM (optional) 25–50 μL/mouse (if 1:1 in 50–100 μL total) On ice; step-by-step on the Matrigel card[2][3] T75 flask or 10-cm dish See expansion table (1 mouse ≥1 flask; 10 ≥6) Harvest at 85–90% confluence Tuberculin syringe 26–27G 1 per mouse + spares Dead space can be ~70 μL; prepare 40–50% extra mix[3] Suture or tissue glue, sterile instruments Per surgical n Autoclave; clip #4 nipple area Caliper Shared for the study Volume ≈ L × W² / 2 Watch the JoVE video first. No anesthetic doses on this page. Do not proceed without IACUC and surgical training. Leak or no bleb = failed inoculum.[2][3]
Check Pass If fail Landmark Female #4 nipple (caudal pair); clip to midline[2] Stop if the landmark is wrong—do not inject blindly Approach Small incision to expose the pad, or slow percutaneous inject (SOP / JoVE) Large incision or ongoing bleed → hemostasis, then decide Needle 26–27G tuberculin; clear air bubbles (Cheng video also uses 25G)[3] Clog: ice the mix—Matrigel may have set Needle dwell if matrix used Wait 10–15 s after the push for the matrix to stiffen, then withdraw to limit leak[3] Immediate withdraw + leak: log as failed inject Inject Slow push until a slight bleb; avoid muscle and peritoneum Leak / no bleb: exclude or reinject only if the protocol allows Close / recover Suture or glue; warm until righting reflex; analgesia per IACUC Prolonged recovery or respiratory depression → humane endpoint / vet SOP - 06[Monitor] Caliper 2–3×/week: volume ≈ L × W² / 2; log weight. Fields on the monitor template. Apply humane endpoints for ulceration or sharp weight loss.
Measure 2–3×/week. Volume ≈ L × W² / 2. Enrollment volume and dosing route live in the IACUC protocol—not invented here.
Field How to log Date / day post-inject D0 = inoculation day Mouse / cage / arm Lock IDs after randomization L, W (mm) Same operator and caliper; longest axis Volume (mm³) L × W² / 2 Body weight (g) Same day as caliper Ulcer / activity / breathing Y/N + brief note; stop if endpoint hit Map to flowchart nodes. Welfare issues follow the IACUC SOP—do not improvise doses from this page.
Sign Likely cause Action Clumps / clogged needle Incomplete digest; Matrigel warmed Gentle triturate or filter; ice the tube; discard set matrix Viability <90% Overconfluence, over-trypsin, too long off ice Do not inject; reculture in log phase and recount Leak / no bleb Volume too large, too fast, wrong plane Log as failed inject; reduce volume and slow the push No palpable tumor by week 2 Missed inject, too few cells, late passage, strain mismatch Audit records; QC before raising dose toward the literature ceiling Ulcer, sharp weight loss, distress Humane endpoint Euthanize per protocol immediately—do not delay for a time point - 07[Optional therapy] Randomize at a set volume (e.g. 100–150 mm³); route and vehicle live in the protocol.
- 08[Optional imaging] Validate Luc/GFP vs parental (Jenkins 2005).
- 09[Endpoint] Weigh/process the primary; optional lung/nodes. Archive passage, inoculum, and lot.
- 10[QC] Fix strain, sex, age, passage, and inoculum; independent repeats. Troubleshoot before changing cell number.
Reagents / materials
| Item | Role | Conc. / dose |
|---|---|---|
| MDA-MB-231 cells (MC-h133) | Orthotopic inoculum | 约 1×10⁶ / 50–100 μL（本 SOP）；保守备瓶按 2×10⁶ |
| Fetal bovine serum (FBS) | 10% complete medium | 常规培养优先 MC100 |
| Matrigel (optional) | Engraftment aid | 1:1 时约 25–50 μL/只；或 25% 基质 |
| 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
Tumor volume curve, body weight, primary tumor weight/pathology, optional lung/LN mets, BLI.
References (PubMed)
- [1]PMID 2297709 — Tumorigenicity and metastasis of human breast carcinoma cell lines in nude mice. Cancer Res (1990)
- [2]PMID 25742185 — Orthotopic injection of breast cancer cells into the mammary fat pad of mice to study tumor growth. J Vis Exp (2015)
- [3]PMID 32250353 — Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model. J Vis Exp (2020)
- [4]PMID 15987449 — Bioluminescent human breast cancer cell lines that permit rapid and sensitive in vivo detection of mammary tumors and multiple metastases in immune deficient mice. Breast Cancer Res (2005)
- [5]PMID 4412247 — Breast tumor cell lines from pleural effusions. J Natl Cancer Inst (1974)
- [6]PMID 730202 — Long-term human breast carcinoma cell lines of metastatic origin: preliminary characterization. In Vitro (1978)
Disclaimer: RUO; IACUC required. Optimize by strain and pilot. Red tags mark weak or non-metastatic parental endpoints.