Use cases
Human bladder TCC T24 s.c. CDX. This SOP uses Ramakrishnan: 5×10⁶ in 100 μL HBSS:Matrigel 50:50, nude. That paper’s HT1376 is 2×10⁶ and UM-UC-3 is 3×10⁶—neither is T24. Not intramural/intravesical orthotopic bladder. Do not copy 5637’s 3×10⁶ / 0.2 mL PBS or MDA-MB-231’s 2×10⁶ / 100 μL.
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
This page is local s.c. CDX only. Orthotopic bladder is another protocol. Do not copy other-line doses from the same paper.
Does the workflow match?
s.c. screens share this skeleton; bladder orthotopic is another protocol.
| Use | vs foolproof SOP | Differences vs core SOP |
|---|---|---|
| s.c. screen | Same core SOP | This page’s Ramakrishnan 5×10⁶ / 100 μL 50:50. |
| Orthotopic bladder | Other route | Parental dose not locked; do not copy the s.c. gel mix. |
Host spec: sex × strain
Ramakrishnan used nude. RUO.
| Indication / context | Sex | Strain / host | Notes |
|---|---|---|---|
| This SOP default: s.c. screen | Not stressed in that paper; per IACUC | nude | 5×10⁶ in 100 μL HBSS:Matrigel 50:50[1]. |
In one line
IACUC → expand → s.c. inject → caliper → optional therapy → 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 (Ramakrishnan：5×10⁶ / 100 μL HBSS:Matrigel 50:50).
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 + nude quarantine
- STR/mycoplasma OK; expand parental T24
- Count (≥90% viable)
- 5×10⁶ in 100 μL HBSS:Matrigel 50:50
- s.c. bleb
- Caliper + weight
- 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-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 5e+6 规划; dead-space ×1.2. Do not reuse MDA-MB-231 orthotopic 2×10⁶ math.
| Animals | T75 flasks |
|---|---|
| 1 | 2 |
| 2 | 3 |
| 3 | 4 |
| 4 | 5 |
| 5 | 6 |
| 6 | 8 |
| 7 | 9 |
| 8 | 10 |
| 9 | 11 |
| 10 | 12 |
Inoculum recipe
This SOP dose string: Ramakrishnan：5×10⁶ / 100 μL HBSS:Matrigel 50:50. Batch ×1.2 for dead space; load one syringe per animal.[1]
| Item | This SOP | Notes |
|---|---|---|
| Cells / volume | Ramakrishnan：5×10⁶ / 100 μL HBSS:Matrigel 50:50 | 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 (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 | Local bleb; no ongoing leak; active after recovery |
| Follow-up window | s.c. tumor volume/weight, 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
Rows are this protocol’s references only—other lines’ Methods numbers are not copied in. Open the PMID.
- 01[Ethics] IACUC; immunodeficient mice. RUO. Ramakrishnan does not give inoculum anesthetic mg/%; immobilize per IACUC.
- 02[Cells] T24 (MC-h208) parental. STR/mycoplasma-qualified. Fogh: nude tumorigenicity panel[2], not an inoculum number.
- 03[Suspension] This SOP 5×10⁶ in 100 μL HBSS:Matrigel 50:50 (Ramakrishnan)[1]. That paper’s HT1376 2×10⁶ and UM-UC-3 3×10⁶ are not T24. Do not default to 5637’s 3×10⁶ / 0.2 mL PBS or 231’s 2×10⁶ / 100 μL.
This SOP dose string: Ramakrishnan：5×10⁶ / 100 μL HBSS:Matrigel 50:50. Batch ×1.2 for dead space; load one syringe per animal.[1]
Item This SOP Notes Cells / volume Ramakrishnan：5×10⁶ / 100 μL HBSS:Matrigel 50:50 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 - 04[Inject] s.c. bleb.
- 05[Monitor] Volume formula from the IACUC SOP. That paper followed T24 for a long window (~86 days)—endpoint per IACUC.
- 06[Endpoint] Tumor pathology. Mets are not an s.c. success criterion.
Reagents / materials
| Item | Role | Conc. / dose |
|---|---|---|
| T24 cells (MC-h208) | Inoculum | Ramakrishnan：5×10⁶ / 100 μL HBSS:Matrigel 50:50 |
| Matrigel (commonly used) | Engraftment aid | 与 HBSS 1:1（50:50），终体积 100 μL（Ramakrishnan） |
| Anesthetic / analgesic | Surgery & welfare | Ramakrishnan 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, body weight, 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.