Use cases
Adriamycin-resistant ER+ MCF-7/ADR abdominal-wall s.c. CDX. This SOP uses Cell Death Dis 3′tRF-AlaAGC: 5×10⁶ cells/mL suspension, 200 μL/mouse (1×10⁶/mouse) into abdominal wall s.c., ♀ Balb/c nude (4–6 wk); that paper explicitly established MCF-7/ADR s.c. xenografts. In vitro 250 ng/mL Adriamycin selection—no mandatory drug at inject. Not parental MCF-7 estrogen-pellet protocol. Do not copy 231’s 2×10⁶ / 100 μL.
Catalog: MC-h253 · Product modeling page
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 locks abdominal-wall s.c. CDX (1×10⁶ in 200 μL). Mammary fat pad orthotopic not locked.
Does the workflow match?
Abdominal wall s.c. 1×10⁶ in 200 μL; ≠ MCF-7 estrogen-pellet flank.
| Use | vs foolproof SOP | Differences vs core SOP |
|---|---|---|
| Abdominal-wall s.c. CDX | Same core SOP | This page’s 1×10⁶ in 200 μL. |
| Fat pad orthotopic | Other route | Parental dose not locked. |
Host spec: sex × strain
Paper used 4–6 wk ♀ Balb/c nude. RUO.
| Indication / context | Sex | Strain / host | Notes |
|---|---|---|---|
| This SOP default: abdominal wall s.c. | Female | Balb/c nude | 1×10⁶ in 200 μL[1]. |
In one line
IACUC → expand resistant line → 1×10⁶ in 200 μL → abdominal wall s.c. inject → caliper → 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 (Cell Death Dis：1×10⁶ in 200 μL 腹壁皮下，♀ Balb/c nude).
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
- Expand MCF-7/ADR
- 1×10⁶ in 200 μL
- Abdominal wall s.c.
- Caliper
- Endpoint
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-h253 datasheet for medium. Matrigel only if the compound card lists it. Anesthetic: compound card/PMID; IACUC may override.[1]
| Item | Per mouse / study | Notes |
|---|---|---|
| MCF-7/Adr cells MC-h253 | 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: Cell Death Dis：1×10⁶ in 200 μL 腹壁皮下，♀ Balb/c nude. Batch ×1.2 for dead space; load one syringe per animal.[1]
| Item | This SOP | Notes |
|---|---|---|
| Cells / volume | Cell Death Dis：1×10⁶ in 200 μL 腹壁皮下，♀ Balb/c nude | 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 (MCF-7/Adr / MC-h253)
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-h253). Feed before harvest as usual for this line. |
| Doubling time | ~34 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, resistance-related IHC/flow. |
| 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.
| Paper | Year | Journal | How to use |
|---|---|---|---|
| Cell Death Dis 2024[1] | ♀ Balb/c nude abdominal wall | 1×10⁶ in 200 μL | This SOP default |
- 01[Ethics] IACUC; immunodeficient mice. RUO.
SKUs link to catalog items. Follow the MC-h253 datasheet for medium. Matrigel only if the compound card lists it. Anesthetic: compound card/PMID; IACUC may override.[1]
Item Per mouse / study Notes MCF-7/Adr cells MC-h253 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 - 02[Cells] MCF-7/Adr (MC-h253) Adriamycin-resistant line; maintain 250 ng/mL Adriamycin in vitro per vendor/paper[1]. STR/mycoplasma-qualified. ER+.
- 03[Suspension] Log-phase cells to 5×10⁶/mL; inject 200 μL/mouse (1×10⁶/mouse)[1]. Do not copy 231’s 2×10⁶ / 100 μL.
- 04[Inject] Abdominal wall s.c.[1]; randomize at ~80 mm³ (paper gave tail-vein agomir).
This SOP dose string: Cell Death Dis：1×10⁶ in 200 μL 腹壁皮下，♀ Balb/c nude. Batch ×1.2 for dead space; load one syringe per animal.[1]
Item This SOP Notes Cells / volume Cell Death Dis：1×10⁶ in 200 μL 腹壁皮下，♀ Balb/c nude 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] Caliper/weight every 3 days.
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 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 - 06[Endpoint] Per IACUC.
SKUs link to catalog items. Follow the MC-h253 datasheet for medium. Matrigel only if the compound card lists it. Anesthetic: compound card/PMID; IACUC may override.[1]
Item Per mouse / study Notes MCF-7/Adr cells MC-h253 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
Reagents / materials
| Item | Role | Conc. / dose |
|---|---|---|
| MCF-7/Adr cells (MC-h253) | Inoculum | Cell Death Dis：1×10⁶ in 200 μL 腹壁皮下，♀ Balb/c nude |
| Anesthetic / analgesic | Surgery & welfare | 皮下未给接种麻醉 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, resistance-related IHC/flow.
References (PubMed)
- [1]PMID 39080676 — tRNA-derived fragment 3'tRF-AlaAGC modulates cell chemoresistance and macrophage polarization in breast cancer. Cell Death Dis (2024)
Disclaimer: RUO; IACUC required. Optimize by strain and pilot. Red tags mark weak or non-metastatic parental endpoints.