Use cases
Microscope-guided unilateral dorsal-lobe (DP) injection of parental LNCaP for an AR+ orthotopic tumor. This SOP uses Liu: 1×10⁶ in 20 μL PBS + 50% Matrigel; 3% isoflurane at 0.8 L/min induction. Sato: same cell number orthotopic, SCID take 89%, 12-week nodes 100% and lung micromets 40% (n=10). Not PC-3 anterior-lobe 50 μL no-gel, and not 231’s 2×10⁶ / 100 μL. Wang is fragment SOI, not this needle.
Catalog: MC-h125 · 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 dorsal-lobe orthotopic growth and Sato’s spread pattern. Flank caliper, SOI fragments, and C4-2 bone models are not this needle.
| Use | Fit | Primary readout | Notes |
|---|---|---|---|
| Dorsal-lobe orthotopic ± nodal/lung spread | Recommended | Primary (US), PSA, node/lung pathology[1][2] | Liu: nude unilateral DP, 1×10⁶ / 20 μL 50% gel, 65% take at 8 weeks (13/20)[1]. Sato: SCID orthotopic 89%; 12-week retroperitoneal/mediastinal nodes 100%, lung micromets 40%[2]. |
| Surgical orthotopic implantation of s.c. fragments (SOI) | Caution | That paper: nodes 61%, lung 44% | Wang: intact tissue sewn onto ventral-lateral lobes—not a 20 μL cell suspension[3]. Liu argues s.c. fragments do not represent the orthotopic microenvironment. |
| Flank caliper / PC-3 anterior lobe / tibia | Not recommended | — | s.c. is the other protocol. Do not copy PC-3 anterior-lobe 50 μL no-gel. Bone mets default to C4-2, not parental[4]. |
Does the workflow match?
Dorsal-lobe orthotopic shares this microscope-inject skeleton; SOI and s.c. are other protocols.
| Use | vs foolproof SOP | Differences vs core SOP |
|---|---|---|
| Dorsal-lobe orthotopic | Same core SOP | This page’s Liu 1×10⁶ / 20 μL. |
| SOI fragment | Other route | Grow s.c. first, then sew prostate—not a microinject. |
| s.c. CDX | Other route | Lim 0.25 mL gel, not 20 μL into the dorsal lobe. |
Host spec: sex × strain
Liu defaults to male BALB/c nude; Sato shows higher orthotopic take in SCID than nude. RUO.
| Indication / context | Sex | Strain / host | Notes |
|---|---|---|---|
| This SOP default: dorsal-lobe orthotopic | Male | BALB/c nude; Liu: purchased at 5 wk, injected after 3 wk acclimation (~8 wk) | 1×10⁶ / 20 μL 50% gel; 3% isoflurane 0.8 L/min[1]. |
| SCID high-spread contrast | Male | SCID (Sato) | Orthotopic 1×10⁶, take 89%; 12-week nodes 100%, lung micromets 40%. Abstract does not state μL[2]. |
In one line
IACUC → expand → microscope-guided DP inject → US/PSA follow-up → multi-organ 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 (Liu：1×10⁶ / 20 μL PBS + 50% Matrigel（单侧背叶）。Sato 对照 1×10⁶ 原位（摘要未写 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 + male nude/SCID quarantine
- STR OK; expand parental LNCaP
- 1×10⁶ / 20 μL PBS + 50% Matrigel
- 3% isoflurane, 0.8 L/min induction (Liu)
- Lower transverse incision; bladder stays in; cotton-stabilize SV to expose DP
- Hamilton 33G slow inject to a small blister
- US follow-up; weight/urinary signs
- Endpoint: prostate + node/lung 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-h125 datasheet for medium. Matrigel only if the compound card lists it. Anesthetic: compound card/PMID; IACUC may override.[1]
| Item | Per mouse / study | Notes |
|---|---|---|
| LNCaP cells MC-h125 | 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 |
Expansion cases: mice vs T75
按本协议出现的最高细胞数 1e+6 规划; dead-space ×1.2. Do not reuse MDA-MB-231 orthotopic 2×10⁶ math.
| Animals | T75 flasks |
|---|---|
| 1 | 1 |
| 2 | 1 |
| 3 | 1 |
| 4 | 1 |
| 5 | 2 |
| 6 | 2 |
| 7 | 2 |
| 8 | 2 |
| 9 | 3 |
| 10 | 3 |
Inoculum recipe
This SOP dose string: Liu：1×10⁶ / 20 μL PBS + 50% Matrigel（单侧背叶）。Sato 对照 1×10⁶ 原位（摘要未写 μL）. Batch ×1.2 for dead space; load one syringe per animal.[1]
| Item | This SOP | Notes |
|---|---|---|
| Cells / volume | Liu：1×10⁶ / 20 μL PBS + 50% Matrigel（单侧背叶）。Sato 对照 1×10⁶ 原位（摘要未写 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
Follow published parameters for this line—do not copy other lines. Anesthetic: compound card/PMID; IACUC may override.
| Check | Pass | If fail |
|---|---|---|
| Route | Match the name/IACUC SOP | Unsure: read the PMID; do not guess |
| Air / clumps | No air; singles on the scope | Clumped: do not inject |
Culture card (LNCaP / MC-h125)
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-h125). 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 | Route checks passed; active after recovery |
| Follow-up window | Primary (US), serum PSA, node/lung pathology, weight. |
| 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
Often no caliper volumes on this route. Weight and IACUC endpoints outrank the image.
| 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 |
| Readout | Primary (US), serum PSA, node/lung pathology, weight. |
Literature case comparison
T75 is planned to Liu/Sato 1×10⁶. 20 μL is not 0.25 mL and not PC-3’s 50 μL.
| Paper | Year | Journal | How to use |
|---|---|---|---|
| Liu 2022[1] | Nude unilateral DP (microscope) | 1×10⁶ / 20 μL PBS + 50% gel | 3% isoflurane 0.8 L/min. 65% take at 8 weeks. 33G Hamilton |
| Sato 1997[2] | SCID orthotopic prostate | 1×10⁶ (abstract: no μL) | Take 89%; 12-week nodes 100%, lung micromets 40% (n=10). PSA nadir ~d4 after castration |
| Wang 1999[3] | Contrast: SOI fragments | Ventral-lateral tissue pieces | 18/20 take; nodes 61%, lung 44%. Not a 20 μL suspension |
| Thalmann 2000[4] | Contrast: C4-2 bone mets | Castrated hosts | Derivative ≠ parental orthotopic SOP |
| Stephenson 1992[5] | LNCaP-FGC ortho vs s.c. | JNCI | Without gel, took only in prostate. No cell number in the abstract; do not copy PC-3M mets rates |
| Horoszewicz 1983[6] | Line establishment | Cancer Res | Identity, not an orthotopic volume |
| Lim 1993[7] | Contrast: s.c. + 0.25 mL gel | 1×10⁶ | Do not copy the volume across routes |
- 01[Ethics] IACUC; male nude/SCID SPF. RUO. Liu: 3% isoflurane induction at 0.8 L/min; keep warm intra-op. IACUC may override. Write weight and urinary endpoints. Peritoneal leak creates artifactual implants.
- 02[Cells] LNCaP (MC-h125) parental/FGC—not C4-2 or LNCaP-LN3. This SOP 1×10⁶ in 20 μL PBS + 50% Matrigel (Liu)[1]. Sato also used 1×10⁶ orthotopic; the abstract does not state μL[2]. Stephenson’s abstract has no parental cell number—do not invent one[5]. Do not copy PC-3 anterior-lobe 50 μL no-gel or 231’s 100 μL. Culture: datasheet first; Liu used DMEM/F12 + 10% FBS, harvest at ~80% confluence.
- 03[Surgery] ~1.5 cm lower transverse incision. Leave the bladder in (externalizing can stretch the pubourethral ligament and cause incontinence). Cotton-ball the SV posteriorly to expose DP; do not rupture the SV with microforceps. Hamilton 33G sharp bevel, roughly parallel to the DP long axis; slow inject to a small blister; cotton pressure on withdrawal. Not ventral lobe, not anterior lobe, not 0.25 mL s.c.
- 04
- 05[Endpoint] Prostate, nodes, lung. Sato n=10: nodes 100%, lung micromets 40%[2]. Peritoneal leak is not cascade success. Bone mets are not a parental default endpoint.
Reagents / materials
| Item | Role | Conc. / dose |
|---|---|---|
| LNCaP cells (MC-h125) | Inoculum | Liu：1×10⁶ / 20 μL PBS + 50% Matrigel（单侧背叶）。Sato 对照 1×10⁶ 原位（摘要未写 μL） |
| Matrigel (commonly used) | Engraftment aid | Liu 50%，总注射 20 μL。不是 Lim 皮下 0.25 mL |
| Anesthetic / analgesic | Surgery & welfare | Liu：3% 异氟烷诱导，0.8 L/min。Sato 未给麻醉数字。批件可覆盖 |
| Test drug / vehicle (optional) | Treatment arm | 按药理方案 |
Readouts
Primary (US), serum PSA, node/lung pathology, weight.
References (PubMed)
- [1]PMID 35168543 — Establishment of an orthotopic prostate cancer xenograft mouse model using microscope-guided orthotopic injection of LNCaP cells into the dorsal lobe of the mouse prostate. BMC Cancer (2022)
- [2]PMID 9108464 — A metastatic and androgen-sensitive human prostate cancer model using intraprostatic inoculation of LNCaP cells in SCID mice. Cancer Res (1997)
- [3]PMID 10334107 — High-malignancy orthotopic nude mouse model of human prostate cancer LNCaP. Prostate (1999)
- [4]PMID 10881018 — LNCaP progression model of human prostate cancer: androgen-independence and osseous metastasis. Prostate (2000)
- [5]PMID 1378502 — Metastatic model for human prostate cancer using orthotopic implantation in nude mice. J Natl Cancer Inst (1992)
- [6]PMID 6831420 — LNCaP model of human prostatic carcinoma. Cancer Res (1983)
- [7]PMID 7681204 — Growth of an androgen-sensitive human prostate cancer cell line, LNCaP, in nude mice. Prostate (1993)
Disclaimer: RUO; IACUC required. Optimize by strain and pilot. Red tags mark weak or non-metastatic parental endpoints.