Use cases
Intramural or intravesical bladder for local growth. Parental T24 default bladder cell number is not locked. Do not copy s.c. 5×10⁶ / 100 μL 50:50 or 5637.
Catalog: MC-h208 · 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
Bladder dose not locked. Do not copy the s.c. gel mix.
| Use | Fit | Primary readout | Notes |
|---|---|---|---|
| Intramural / intravesical (pilot) | Caution | Bladder burden, pathology | Cell number/volume from the IACUC pilot only. |
| s.c. CDX | Not recommended | — | Use the s.c. protocol. |
Does the workflow match?
Bladder orthotopic and s.c. are different paths.
| Use | vs foolproof SOP | Differences vs core SOP |
|---|---|---|
| Orthotopic bladder | Same core SOP | This page; dose not locked. |
| s.c. CDX | Other route | Ramakrishnan 5×10⁶ / 100 μL 50:50, not bladder. |
Host spec: sex × strain
Immunodeficient mice; sex per IACUC. RUO.
| Indication / context | Sex | Strain / host | Notes |
|---|---|---|---|
| Bladder (pilot) | Per IACUC | Immunodeficient | Dose from the IACUC SOP only. |
In one line
Bladder inject → monitor → endpoint.
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 (亲本膀胱剂量未锁——批件预实验；勿抄皮下).
4. After
Follow the monitor log; stop at endpoints.
Protocol overview
Scenario-specific flowchart (core engraftment skeleton with host/therapy or imaging/readout changes).
- Expand parental T24
- Pilot per IACUC
- Bladder inject
- Monitor
- Endpoint 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-h208 datasheet for medium. Matrigel only if the compound card lists it. Anesthetic: compound card/PMID; IACUC may override.[1]
| Item | Per mouse / study | Notes |
|---|---|---|
| T24 cells MC-h208 | 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: 亲本膀胱剂量未锁——批件预实验；勿抄皮下. Batch ×1.2 for dead space; load one syringe per animal.[1]
| Item | This SOP | Notes |
|---|---|---|
| Cells / volume | 亲本膀胱剂量未锁——批件预实验；勿抄皮下 | 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 (T24 / MC-h208)
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-h208). Feed before harvest as usual for this line. |
| Doubling time | ~19 hours (Cellosaurus; not a measured lot) |
| 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 | Bladder burden, 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
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 | Bladder burden, pathology. |
Literature case comparison
T75 planned to 1×10⁶ is a bladder methods starting point only. Parental bladder dose not locked.
| Paper | Year | Journal | How to use |
|---|---|---|---|
| This page | Orthotopic bladder | Parental dose not locked | Do not copy s.c. 5×10⁶ / 100 μL 50:50 |
| Ramakrishnan 2018[1] | Contrast: s.c. | 5×10⁶ in 100 μL 50:50 | Do not copy across routes |
- 01[Ethics] IACUC; lower-urinary-tract endpoints. RUO.
- 02[Cells] T24 (MC-h208). Parental bladder dose not locked. Do not copy s.c. 5×10⁶ / 100 μL 50:50.
- 03[Inject] Intramural or intravesical per validated SOP.
- 04[Endpoint] Bladder pathology.
Reagents / materials
| Item | Role | Conc. / dose |
|---|---|---|
| T24 cells (MC-h208) | Inoculum | 亲本膀胱剂量未锁——批件预实验；勿抄皮下 |
| Anesthetic / analgesic | Surgery & welfare | 仅按批件 |
| Test drug / vehicle (optional) | Treatment arm | 按药理方案 |
Readouts
Bladder burden, pathology.
References (PubMed)
- [1]PMID 30038946 — Transcriptional changes associated with in vivo growth of muscle-invasive bladder cancer cell lines in nude mice. Am J Clin Exp Urol (2018)
- [2]PMID 327080 — One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice. J Natl Cancer Inst (1977)
Disclaimer: RUO; IACUC required. Optimize by strain and pilot. Red tags mark weak or non-metastatic parental endpoints.