Use cases
AR+/PSA+ LNCaP flank s.c. CDX usually needs Matrigel for endocrine/castration pharmacology. This SOP uses Lim: 1×10⁶ + 0.25 mL Matrigel. Sato contrast: same cell number s.c. (100% take in SCID); that abstract does not state μL. Not orthotopic prostate. Do not copy MDA-MB-231’s 2×10⁶ / 100 μL, and do not default 0.25 mL gel to 100 μL.
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
This page is local s.c. CDX only. Orthotopic spread is the other protocol. Stephenson’s no-gel s.c. did not take; C4-2/LNCaP-LN3 are not parental.
| Use | Fit | Primary readout | Notes |
|---|---|---|---|
| AR+ endocrine / castration screen | Recommended | s.c. volume, serum PSA, weight[1] | Lim: 1×10⁶ in 0.25 mL Matrigel s.c.; ~88% palpable by 12 weeks; bilateral orchiectomy shrinks tumors and stabilizes PSA[1]. |
| SCID high-take s.c. (contrast) | Suitable | Same caliper; that paper’s s.c. had no nodal/distant mets | Sato: 1×10⁶ s.c., SCID 65/65 (100%), athymic 80%; abstract does not state volume/gel. Mets are not an s.c. success criterion[2]. |
| Orthotopic prostate / bone mets | Not recommended | — | Use the orthotopic protocol. Bone colonization is C4-2-class derivatives (Thalmann), not parental LNCaP[3]. |
Does the workflow match?
Endocrine screens share this s.c. skeleton; dorsal-lobe orthotopic is another protocol.
| Use | vs foolproof SOP | Differences vs core SOP |
|---|---|---|
| AR+ s.c. screen | Same core SOP | This page’s Lim 1×10⁶ / 0.25 mL gel. |
| Dorsal-lobe orthotopic | Other route | Liu 1×10⁶ / 20 μL 50% gel, not 0.25 mL s.c. |
Host spec: sex × strain × androgen
Human LNCaP uses immunodeficient males. Horoszewicz: males take earlier and more often; take frequency tracks serum androgen. RUO.
| Indication / context | Sex | Strain / host | Notes |
|---|---|---|---|
| This SOP default: s.c. endocrine screen | Male (testis-intact) | Nude (Lim) or SCID (Sato higher take) | Lim: 1×10⁶ in 0.25 mL Matrigel[1]. Castration is an experimental arm, not the default inoculum condition. |
| Post-castration regression / CRPC | Male → castrated | Same strain | Lim: bilateral orchiectomy shrinks tumors and stabilizes PSA[1]. Sato: PSA falls ~65% after castration; s.c. nadir ~day 7[2]. C4-2 is the derivative that takes in castrated hosts[3]. |
| Female | Not the default | — | Horoszewicz: females take later and less often; once established, growth rate is independent of sex[4]. |
In one line
IACUC → expand → s.c. (Lim: large-volume Matrigel) → caliper/PSA → optional therapy or castration → 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 (Lim：1×10⁶ + 0.25 mL Matrigel。Sato 对照 1×10⁶ 皮下（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/mycoplasma OK; expand parental LNCaP (FGC; do not mix C4-2)
- Count (≥90% viable)
- ~1×10⁶ + 0.25 mL Matrigel (Lim)
- Flank s.c. bleb
- Weekly caliper + weight; optional serum PSA
- Randomize at set volume or castrate
- Endpoint tumor 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: Lim：1×10⁶ + 0.25 mL Matrigel。Sato 对照 1×10⁶ 皮下（摘要未写 μL）. Batch ×1.2 for dead space; load one syringe per animal.[1]
| Item | This SOP | Notes |
|---|---|---|
| Cells / volume | Lim：1×10⁶ + 0.25 mL Matrigel。Sato 对照 1×10⁶ 皮下（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
Flank s.c. bleb; avoid muscle. Anesthetic: compound card/PMID; IACUC may override.
| Check | Pass | If fail |
|---|---|---|
| Site | Flank s.c. bleb | Intramuscular: log as failed |
| Needle | Often 25–27G; follow the paper | Do not assume orthotopic tuberculin dead space |
| Matrix | Only if this protocol’s compound card lists Matrigel | Never put Matrigel on an i.v. SOP |
| 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 | Local bleb; no ongoing leak; active after recovery |
| Follow-up window | s.c. tumor volume/weight, serum PSA, body weight, pathology. |
| 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
T75 is planned to Lim/Sato 1×10⁶. 0.25 mL gel is not 100 μL; s.c. is not dorsal lobe.
| Paper | Year | Journal | How to use |
|---|---|---|---|
| Lim 1993[1] | Nude s.c. + Matrigel | 1×10⁶ / 0.25 mL gel | ~88% palpable by 12 weeks; castration shrinks. Not 100 μL |
| Sato 1997[2] | SCID/nude s.c. | 1×10⁶ (abstract: no μL/gel) | SCID 100%; no s.c. mets. PSA ~65% drop after castration; nadir ~d7 |
| Thalmann 2000[3] | Contrast: C4-2 progression | Castrated hosts + bone mets | Derivative ≠ parental s.c. SOP |
| Horoszewicz 1983[4] | LNCaP establishment | Cancer Res | Males take more often; not this SOP’s cell number |
| van Steenbrugge 1989[5] | FGC and subline overview | Urol Res | FGC androgen-dependent; LNO etc. can be independent |
| Stephenson 1992[6] | No gel: LNCaP took only in prostate | JNCI | Not a gelled s.c. number; do not copy PC-3M mets rates |
| Pretlow 1991[7] | Matrigel take principle | PC-3 needed 25,000-fold fewer cells | Not an LNCaP dose |
- 01[Ethics] IACUC; male immunodeficient SPF. RUO. Lim/Sato s.c. do not give anesthetic mg/%; immobilize per IACUC if needed. Write ulcer and weight endpoints.
- 02[Cells] LNCaP (MC-h125), STR/mycoplasma-qualified. Horoszewicz: lymph-node-met origin, AR+, in-vitro doubling ~60 h[4]. ATCC usually ships clone FGC (CRL-1740). van Steenbrugge: FGC is androgen-dependent; LNO/R sublines can be independent—do not mix[5]. Log phase, ≥90% viable. Datasheet first; Liu’s orthotopic paper used DMEM/F12 + 10% FBS. Cellosaurus doubling is multi-source ~34–43 h (ATCC ~34 h)—not a measured lot.
- 03[Suspension] This SOP ~1×10⁶ + 0.25 mL Matrigel (Lim)[1]. Do not default to 100 μL or MDA-MB-231’s 2×10⁶. Sato also used 1×10⁶ s.c.; the abstract does not state volume/gel[2]. Stephenson: without gel, LNCaP took only in the prostate, not s.c.—no-gel s.c. is not this SOP[6]. Pretlow’s 25,000-fold reduction is a PC-3 number, not LNCaP[7].
- 04[Inject] Flank s.c. bleb; avoid muscle. Large gel volume—push slowly to limit leak.
This SOP dose string: Lim：1×10⁶ + 0.25 mL Matrigel。Sato 对照 1×10⁶ 皮下（摘要未写 μL）. Batch ×1.2 for dead space; load one syringe per animal.[1]
Item This SOP Notes Cells / volume Lim：1×10⁶ + 0.25 mL Matrigel。Sato 对照 1×10⁶ 皮下（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 Flank s.c. bleb; avoid muscle. Anesthetic: compound card/PMID; IACUC may override.
Check Pass If fail Site Flank s.c. bleb Intramuscular: log as failed Needle Often 25–27G; follow the paper Do not assume orthotopic tuberculin dead space Matrix Only if this protocol’s compound card lists Matrigel Never put Matrigel on an i.v. SOP Air / clumps No air; singles on the scope Clumped: do not inject - 05[Monitor] Volume ≈ L×W²/2 (this site’s log). Lim: PSA correlates with volume/weight[1]. Slower than PC-3; scoring take at 12 weeks is not unusual.
- 06[Endpoint] Tumor pathology. Nodal/lung mets are not an s.c. success criterion (Sato s.c. had none[2]). Castration micrograms/surgery follow IACUC only.
Reagents / materials
| Item | Role | Conc. / dose |
|---|---|---|
| LNCaP cells (MC-h125) | Inoculum | Lim：1×10⁶ + 0.25 mL Matrigel。Sato 对照 1×10⁶ 皮下（摘要未写 μL） |
| Matrigel (commonly used) | Engraftment aid | Lim 0.25 mL。不要默认为 100 μL 或 1:1 小体积 |
| Anesthetic / analgesic | Surgery & welfare | Lim/Sato 皮下未给麻醉 mg/%；需要制动时按批件 |
| Test drug / vehicle (optional) | Treatment arm | 按药理方案 |
Readouts
s.c. tumor volume/weight, serum PSA, body weight, pathology.
References (PubMed)
- [1]PMID 7681204 — Growth of an androgen-sensitive human prostate cancer cell line, LNCaP, in nude mice. Prostate (1993)
- [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 10881018 — LNCaP progression model of human prostate cancer: androgen-independence and osseous metastasis. Prostate (2000)
- [4]PMID 6831420 — LNCaP model of human prostatic carcinoma. Cancer Res (1983)
- [5]PMID 2660395 — The human prostatic carcinoma cell line LNCaP and its derivatives. An overview. Urol Res (1989)
- [6]PMID 1378502 — Metastatic model for human prostate cancer using orthotopic implantation in nude mice. J Natl Cancer Inst (1992)
- [7]PMID 2065335 — Transplantation of human prostatic carcinoma into nude mice in Matrigel. Cancer Res (1991)
Disclaimer: RUO; IACUC required. Optimize by strain and pilot. Red tags mark weak or non-metastatic parental endpoints.