Use cases
Flank s.c. MDA-MB-231 as a caliper-friendly ectopic CDX for early antitumor efficacy. Not a primary metastasis model (s.c. xenografts often fail to metastasize vs orthotopic[1]), but occasional co-graft papers still report early spread—tagged weak, not absolutely non-metastatic.
Catalog: MC-h133 · Product modeling page · In-vitro spheroid
Research use only (RUO)—not clinical care. IACUC approval and SPF housing are required. This page does not give anesthetic/analgesic doses and does not replace the veterinary SOP. Do not proceed without approval. Flank s.c. is not #4 fat-pad surgery.
Use-case overview
Use this page for caliper efficacy; switch to orthotopic or experimental-mets protocols for dissemination—do not reuse this flank SOP as-is.
| Use | Fit | Primary readout | Notes |
|---|---|---|---|
| Caliper efficacy screen | Recommended | s.c. volume/weight, body weight, pathology[1][2][3] | Flank s.c.; female nude, NOD-SCID, or NSG. |
| Take-rate / formulation QC | Suitable | Time-to-take, enrollment-volume repeatability | Same inoculum skeleton; log passage and Matrigel lot. |
| Primary metastasis model (spontaneous or experimental) | Not recommended | Lung/bone/brain colonization is not this SOP | s.c. is a poor mets model[1]; use orthotopic or tail-vein/intracardiac/brain protocols. |
Does the workflow match?
Efficacy and QC share this flank-inoculum skeleton; metastasis endpoints need another protocol.
| Use | vs foolproof SOP | Differences vs core SOP |
|---|---|---|
| Caliper efficacy screen | Same core SOP | This page is the core SOP. |
| Take-rate / formulation QC | Same core SOP | Same inject; follow-up can be shorter. |
| Primary metastasis model | Other route | Orthotopic fat pad or tail vein/intracardiac/intracranial—not flank s.c. |
Host spec: sex × strain × age
BITC used female nudes[2], Saghaeidehkordi NOD-SCID[1], Ring NSG[3]. This SOP defaults to female nudes. Do not mix volume curves across hosts. Quarantine per facility SOP, often ≥3–7 days (BITC: 1 wk acclimation). RUO.
| Indication / context | Sex | Strain / host | Notes |
|---|---|---|---|
| This SOP default: flank growth / efficacy | Female (required) | Nude nu/nu; 6–8 weeks; quarantine ≥3–7 days | Matches BITC 6–7 wk + 1 wk acclimation[2]; ~1–2×10⁶ / 100 μL; pilot n=3–5. |
| NOD-SCID efficacy series | Female | NOD-SCID; 8 weeks | 2×10⁶ in 100 μL DMEM/F12:Matrigel 1:1, right flank, 25G[1]. ~131 mm³ at d26. |
| Faster take on NSG | Female | NSG; 8–10 weeks | Ring: 1×10⁶ / 100 μL, left hind flank, tissue glue[3]. Large by 3 wk—watch endpoints earlier. |
| Male | Not the default | — | All three flank SOPs are female; males need written justification and do not extrapolate cleanly. |
In one line
IACUC → expand cells → flank s.c. (Matrigel 1:1) → caliper → optional therapy → endpoint weight/pathology.
D−1 / D0 timeline
1. D−7 to D−3
SPF quarantine (BITC: 1 wk acclimation after arrival)[[2]]; confirm female/age; IACUC in hand.
2. D−1
Feed cells at 85–90% confluence; weigh; optional flank clip for later caliper (not #4 nipple prep).
3. D0 morning
PBS wash → trypsin → neutralize/spin → serum-free resuspend → count (≥90% viable).
4. D0 mix
Mix 1:1 per recipe + Matrigel card (+ ~20% extra); stay on ice.
5. D0 inject
Tent skin → slow flank s.c. to a bleb → twist or glue. Finish within 30–60 min.
6. D0 after inject
If anesthetized, warm until righting; log dose, side, and passage.
7. From D+3
Caliper + weight 1–2×/week; stop at humane endpoints.
Protocol overview
Scenario-specific flowchart (core engraftment skeleton with host/therapy or imaging/readout changes).
- IACUC + immunodeficient quarantine
- Expand MDA-MB-231 (log passage; T75 on expansion table)
- Day-of count (viability ≥90%)
- Flank subcutaneous inject (Matrigel 1:1, 25G)
- Weekly caliper + body weight
- Optional therapy at set volume
- Endpoint tumor pathology
Novice pack (literature cases)
Flowchart buttons open the matching table. Full pack below, in use order.
Unpack list (flank s.c., plan per mouse)
SKUs link to catalog items. Follow the MC-h133 datasheet; flank papers use DMEM/F12 or DMEM + 10% FBS. This route commonly uses 1:1 Matrigel and a 25G needle—not the orthotopic fat-pad kit.[1][2][3]
| Item | Per mouse / study | Notes |
|---|---|---|
| MDA-MB-231 cells MC-h133 | Start from 1 vial | STR / mycoplasma-qualified; log passage |
| FBS (routine culture) MC100 | 10% in complete medium | Prefer regular grade; HI only if datasheet says so |
| Medium (DMEM/F12 or DMEM) | ~15 mL per T75 | Saghaeidehkordi: DMEM/F12[1]; Ring: DMEM[3]; datasheet may list L-15 |
| Ca²⁺/Mg²⁺-free PBS / DPBS | Wash + resuspend; inject volume on recipe card | Keep inoculum serum-free; BITC used PBS:Matrigel 1:1[2] |
| Trypsin-EDTA (0.05–0.25%) | ~3–5 mL per T75 | Single cells; avoid over-trypsinizing |
| Matrigel / BMM (typical on this route) | 50 μL/mouse (100 μL total at 1:1) | On ice; ratios on the Matrigel card[1][2][3] |
| T75 flask or 10-cm dish | See expansion table (three dose columns) | Harvest at 85–90% confluence |
| 1 mL syringe + 25G needle | 1 set per mouse + spares | 25G to limit shearing[1]; less dead space than tuberculin—prepare ~20% extra |
| Tissue adhesive (optional) | Per puncture | Ring sealed the left-flank puncture with degradable glue[3]; outward needle twist also used[1] |
| Caliper | Shared for the study | This page: volume ≈ L × W² / 2; do not mix with the ellipsoid formula (monitor card) |
Expansion cases: mice vs T75 (three flank doses)
Assumes ~5×10⁶ harvested per log-phase T75 and ~20% extra for a 25G 1 mL syringe (not the orthotopic tuberculin 50% dead-space). Columns = this SOP typical 1×10⁶, typical ceiling 2×10⁶, BITC high-dose arm 5×10⁶/site. A 10-cm dish is close in area. For bilateral flanks, count injection sites (1 mouse bilateral ≈ 2 sites).[1][2][3]
| Injection sites | SOP 1×10⁶ T75 | SOP 2×10⁶ T75 | High-dose 5×10⁶ T75 |
|---|---|---|---|
| 1 | 1 | 1 | 2 |
| 2 | 1 | 1 | 3 |
| 3 | 1 | 2 | 4 |
| 4 | 1 | 2 | 5 |
| 5 | 2 | 3 | 6 |
| 6 | 2 | 3 | 8 |
| 7 | 2 | 4 | 9 |
| 8 | 2 | 4 | 10 |
| 9 | 3 | 5 | 11 |
| 10 | 3 | 5 | 12 |
Inoculum recipe (per mouse + batch)
This SOP typically uses ~1–2×10⁶ in 100 μL at Matrigel 1:1. The high-dose arm matches BITC 2.5–5×10⁶—do not reuse the orthotopic 2×10⁶ × 1.5 flask math. Keep on ice; inject within 30–60 min.[1][2][3]
| Item | This SOP (typical) | Literature ceiling / variant |
|---|---|---|
| Cells / site | ~1–2×10⁶ | Ring 1×10⁶[3]; Saghaeidehkordi 2×10⁶[1]; BITC 2.5–5×10⁶[2] |
| Volume / site | 100 μL | All three use 0.1 mL; Ring mixed 150 μL slurry then injected 100 μL[3] |
| Matrix | Matrigel 1:1 (50 μL cells + 50 μL matrix) | DMEM/F12:Matrigel 1:1[1][3] or PBS:Matrigel 1:1[2] |
| Uni- vs bilateral | One hind flank (right[1] or left hind[3]) | BITC: both flanks above the hind limb, one shot/side[2] |
| Batch multiplier | sites × 1.2 (25G dead space / loss) | Ring mixed 150 μL / injected 100 μL ≈ 50% extra[3] |
| Batch for 10 sites (SOP 2×10⁶) | ~2.4×10⁷ (2×10⁶ × 10 × 1.2) → T75 2×10⁶ column | High-dose 5×10⁶: ~6.0×10⁷ → T75 right column |
| Viability | Trypan blue ≥90% | Do not inject if <90%; reculture |
Matrigel: flank 1:1 (do not copy fat-pad 25%)
The three flank papers all use ~1:1 matrix—not Cheng’s orthotopic 25% BMM. Cold handling follows the product insert; ratios and leak control are from the flank papers only.[1][2][3]
| Item | Practice in this experiment |
|---|---|
| Use it or skip it | This SOP typically uses it, 1:1. All three flank papers include Matrigel[1][2][3]. One choice for the whole study; log the lot. Do not copy the orthotopic 25% mix onto the flank. |
| Thaw and store | Follow the insert: typically −20 °C stock; thaw fully at 4 °C / on ice (often overnight)—do not thaw at room temperature or in a water bath until it gels. Aliquot on ice. Keep on ice while in use. |
| Chill the hardware | Pre-chill tips, tubes, 1 mL syringes/25G needles, PBS or serum-free medium, and the cell pellet. Finish warm steps (trypsin, neutralize, count) before mixing. |
| Mix (this SOP, 1:1) | After counting, resuspend at 2× final density in ice-cold serum-free DMEM/F12 or PBS, then gently mix 1:1 with ice-cold Matrigel (no foam). Final 100 μL/site. Saghaeidehkordi: DMEM/F12:Matrigel 1:1[1]; BITC: PBS:Matrigel 1:1[2]. |
| Ring variant (mix 150, inject 100) | 1×10⁶ cells in 75 μL DMEM/F12 + 75 μL Matrigel (150 μL slurry); inject 100 μL into the left hind flank[3]. The leftover ~50 μL covers loading loss. |
| Load and limit leak | Draw with a pre-chilled syringe; clear bubbles. After the bleb: twist the needle slightly outward before withdrawing[1], or seal the puncture with tissue adhesive[3]. Finish in 30–60 min. If the mix strings, thickens, or clogs, discard the tube. |
Injection checklist (flank s.c., not fat-pad surgery)
Tent the skin and raise a bleb—do not open the #4 fat pad. No anesthetic doses on this page. Do not proceed without IACUC. Leak, no bleb, or intramuscular placement = failed inoculum.[1][2][3]
| Check | Pass | If fail |
|---|---|---|
| Landmark | Hind-flank subcutis; right[1], left hind[3], or both flanks[2] | Stop if the plane is muscle—do not inject blindly |
| Approach | Tent the skin; needle between skin and muscle; a mobile bleb | No bleb or high resistance: withdraw—do not force |
| Needle | 1 mL + 25G (limit shearing)[1] | Clog: ice the mix—Matrigel may have set |
| Leak control | Twist outward after the bleb[1], or seal with tissue glue[3] | Immediate withdraw + leak: log that site as failed |
| Uni- / bilateral | One scheme for the study; bilateral = one shot/side, dose per site[2] | Do not dump a failed site’s dose into the other flank unless the protocol allows |
| Recover | If anesthetized: warm until righting; analgesia per IACUC. If restrained awake: watch then cage | Prolonged recovery or respiratory depression → humane endpoint / vet SOP |
Culture card (MDA-MB-231 / MC-h133)
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 MC-h133 datasheet. Confluence = % of growth area covered by attached cells; if the monolayer is even, that is about the fraction of the inverted-microscope field occupied by cells. Schematic, not a real micrograph. Flank papers harvest exponentially growing cells[2].
| Item | Practice |
|---|---|
| Medium | Datasheet first. Saghaeidehkordi: DMEM/F12 + 10% FBS, 37°C, 5% CO₂[1]. Ring: DMEM + 10% FBS[3]. ATCC classic also uses L-15 + 10% FBS (watch CO₂). |
| 85–90% confluence (split / harvest) | Under the inverted microscope cells occupy ~85–90% of the field: nearly full, with small gaps, no stacking or peeling. MDA-MB-231 is spindle-like, so 90% looks looser than cobblestone epithelia—judge by gaps, do not wait for 100%. ~50% is too sparse for yield; a packed monolayer drops viability and should not be injected. BITC used exponentially growing cells[2]. |
| Feed / split | Medium ≥2×/week; split at 85–90% confluence as above—keep log phase, do not overgrow. BITC: inject exponentially growing cells[2]. |
| Passage | Fix an early–mid window for in-vivo work and log it. |
| Day −1 | Feed; check morphology; expand enough T75s for injection-site n (see expansion table). |
What success looks like (expected, not a guarantee)
Kinetics vary by strain, passage, and dose—pilot first. If off-track, use the troubleshooting table before raising cell number.[1][3]
| Time point | Typical appearance |
|---|---|
| Day 0 | Clear mobile flank bleb; no obvious leak; mouse active |
| ~Day 4 | Visible growth reported with 2×10⁶ in NOD-SCID[1] |
| ~Day 26 | NOD-SCID, 2×10⁶: ~130.9 ± 30 mm³ (that paper’s ellipsoid formula)[1]; efficacy often randomizes at 100–150 mm³[1] |
| ~Week 3 (NSG, 1×10⁶) | Ring WT flank tumors already large (~756 mm³)[3]; faster than the NOD-SCID series—do not compare volumes across hosts |
| Take rate | Parental MDA-MB-231 takes well on the flank, but lots/passage vary; pilot n ≈ 3–5 |
Troubleshooting
Map to flowchart nodes. Welfare issues follow the IACUC SOP—do not improvise doses from this page.
| Sign | Likely cause | Action |
|---|---|---|
| Clumps / clogged needle | Incomplete digest; Matrigel warmed | Gentle triturate or filter; ice the tube; discard set matrix |
| Viability <90% | Overconfluence, over-trypsin, too long off ice | Do not inject; reculture in log phase and recount |
| Leak / no bleb | Volume too large, too fast, skin not tented | Log as failed inject; slow the push; twist or glue |
| Mass fixed, not mobile with skin | Likely intramuscular | Exclude from efficacy; correct the plane in the pilot |
| No palpable tumor by week 2 | Missed inject, too few cells, late passage, strain mismatch | Audit records; QC before raising dose toward the literature ceiling |
| Ulcer, sharp weight loss, distress | Humane endpoint | Euthanize per protocol immediately—do not delay for a time point |
Monitoring log fields (print / copy)
This page defaults to volume ≈ L × W² / 2 (same family as Ring’s W²L/2). Saghaeidehkordi used ¾π × L × W² × 1/8—do not mix formulas in one study. Frequency: twice weekly in that paper[1]; weekly in Ring[3].
| Field | How to log |
|---|---|
| Date / day post-inject | D0 = inoculation day |
| Mouse / cage / arm / side | Lock IDs after randomization; split L/R if bilateral |
| L, W (mm) | Same operator and caliper; longest axis |
| Volume (mm³) | This page: L × W² / 2 |
| Body weight (g) | Same day as caliper |
| Ulcer / activity / breathing | Y/N + brief note; stop if endpoint hit |
Literature case comparison
This SOP uses a mid 1–2×10⁶ dose; the T75 table stocks to the high-dose (right-hand) column. Open PMIDs for the papers.
| Paper | Host | Cells | Volume / matrix / needle | Readout |
|---|---|---|---|---|
| Saghaeidehkordi 2021[1] | ♀ NOD-SCID, 8 wk | 2×10⁶ / right flank | 100 μL DMEM/F12:Matrigel 1:1; 1 mL + 25G; outward twist | Caliper 2×/week; d26 ≈ 131 mm³; enroll 100–150 mm³ |
| Warin / BITC 2010[2] | ♀ nude nu/nu, 6–7 wk, 1 wk acclimation | 2.5–5×10⁶ / flank, bilateral | 0.1 mL PBS:Matrigel 1:1; flanks above hind limb | Caliper + weight; exponential-phase cells |
| Ring 2020[3] | ♀ NSG, 8–10 wk | 1×10⁶ / left hind flank | Mix 150 μL (75+75), inject 100 μL; tissue glue | Weekly caliper; WT ~756 mm³ by 3 wk |
- 01[Ethics/facility] Obtain IACUC approval; quarantine female immunodeficient mice ≥3–7 days under SPF (BITC: 1 wk acclimation[2]). See unpack list and host table. Handle MDA-MB-231 per biosafety. RUO. This page does not replace training or the veterinary SOP.
SKUs link to catalog items. Follow the MC-h133 datasheet; flank papers use DMEM/F12 or DMEM + 10% FBS. This route commonly uses 1:1 Matrigel and a 25G needle—not the orthotopic fat-pad kit.[1][2][3]
Item Per mouse / study Notes MDA-MB-231 cells MC-h133 Start from 1 vial STR / mycoplasma-qualified; log passage FBS (routine culture) MC100 10% in complete medium Prefer regular grade; HI only if datasheet says so Medium (DMEM/F12 or DMEM) ~15 mL per T75 Saghaeidehkordi: DMEM/F12[1]; Ring: DMEM[3]; datasheet may list L-15 Ca²⁺/Mg²⁺-free PBS / DPBS Wash + resuspend; inject volume on recipe card Keep inoculum serum-free; BITC used PBS:Matrigel 1:1[2] Trypsin-EDTA (0.05–0.25%) ~3–5 mL per T75 Single cells; avoid over-trypsinizing Matrigel / BMM (typical on this route) 50 μL/mouse (100 μL total at 1:1) On ice; ratios on the Matrigel card[1][2][3] T75 flask or 10-cm dish See expansion table (three dose columns) Harvest at 85–90% confluence 1 mL syringe + 25G needle 1 set per mouse + spares 25G to limit shearing[1]; less dead space than tuberculin—prepare ~20% extra Tissue adhesive (optional) Per puncture Ring sealed the left-flank puncture with degradable glue[3]; outward needle twist also used[1] Caliper Shared for the study This page: volume ≈ L × W² / 2; do not mix with the ellipsoid formula (monitor card) - 02[Cells / Expand MDA-MB-231 (log passage)] STR/mycoplasma-qualified MC-h133; fixed passage. Feed on D−1; harvest at 85–90% confluence, exponential phase[2]. See culture card and T75 table (1/2/5×10⁶ columns—do not reuse orthotopic flask math).[1][2][3]
Assumes ~5×10⁶ harvested per log-phase T75 and ~20% extra for a 25G 1 mL syringe (not the orthotopic tuberculin 50% dead-space). Columns = this SOP typical 1×10⁶, typical ceiling 2×10⁶, BITC high-dose arm 5×10⁶/site. A 10-cm dish is close in area. For bilateral flanks, count injection sites (1 mouse bilateral ≈ 2 sites).[1][2][3]
Injection sites SOP 1×10⁶ T75 SOP 2×10⁶ T75 High-dose 5×10⁶ T75 1 1 1 2 2 1 1 3 3 1 2 4 4 1 2 5 5 2 3 6 6 2 3 8 7 2 4 9 8 2 4 10 9 3 5 11 10 3 5 12 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.
Click to enlargeLarge gaps; wait one more day Nearly full, small gaps; no stacking No gaps; do not inject Follow the MC-h133 datasheet. Confluence = % of growth area covered by attached cells; if the monolayer is even, that is about the fraction of the inverted-microscope field occupied by cells. Schematic, not a real micrograph. Flank papers harvest exponentially growing cells[2].
Item Practice Medium Datasheet first. Saghaeidehkordi: DMEM/F12 + 10% FBS, 37°C, 5% CO₂[1]. Ring: DMEM + 10% FBS[3]. ATCC classic also uses L-15 + 10% FBS (watch CO₂). 85–90% confluence (split / harvest) Under the inverted microscope cells occupy ~85–90% of the field: nearly full, with small gaps, no stacking or peeling. MDA-MB-231 is spindle-like, so 90% looks looser than cobblestone epithelia—judge by gaps, do not wait for 100%. ~50% is too sparse for yield; a packed monolayer drops viability and should not be injected. BITC used exponentially growing cells[2]. Feed / split Medium ≥2×/week; split at 85–90% confluence as above—keep log phase, do not overgrow. BITC: inject exponentially growing cells[2]. Passage Fix an early–mid window for in-vivo work and log it. Day −1 Feed; check morphology; expand enough T75s for injection-site n (see expansion table). - 03[Harvest] PBS wash, trypsinize to single cells, neutralize, centrifuge; resuspend serum-free or in PBS. Trypan blue ≥90%. Ice; inject within 30–60 min. Batch math on the recipe card (~20% extra).
- 04
- 05[Flank inject] Follow the injection checklist: tent skin, 25G, slow bleb, twist or glue. Do not open the #4 fat pad. Anesthetic/analgesic doses come only from the IACUC SOP.
SKUs link to catalog items. Follow the MC-h133 datasheet; flank papers use DMEM/F12 or DMEM + 10% FBS. This route commonly uses 1:1 Matrigel and a 25G needle—not the orthotopic fat-pad kit.[1][2][3]
Item Per mouse / study Notes MDA-MB-231 cells MC-h133 Start from 1 vial STR / mycoplasma-qualified; log passage FBS (routine culture) MC100 10% in complete medium Prefer regular grade; HI only if datasheet says so Medium (DMEM/F12 or DMEM) ~15 mL per T75 Saghaeidehkordi: DMEM/F12[1]; Ring: DMEM[3]; datasheet may list L-15 Ca²⁺/Mg²⁺-free PBS / DPBS Wash + resuspend; inject volume on recipe card Keep inoculum serum-free; BITC used PBS:Matrigel 1:1[2] Trypsin-EDTA (0.05–0.25%) ~3–5 mL per T75 Single cells; avoid over-trypsinizing Matrigel / BMM (typical on this route) 50 μL/mouse (100 μL total at 1:1) On ice; ratios on the Matrigel card[1][2][3] T75 flask or 10-cm dish See expansion table (three dose columns) Harvest at 85–90% confluence 1 mL syringe + 25G needle 1 set per mouse + spares 25G to limit shearing[1]; less dead space than tuberculin—prepare ~20% extra Tissue adhesive (optional) Per puncture Ring sealed the left-flank puncture with degradable glue[3]; outward needle twist also used[1] Caliper Shared for the study This page: volume ≈ L × W² / 2; do not mix with the ellipsoid formula (monitor card) - 06[Monitor] Caliper 1–2×/week: this page volume ≈ L × W² / 2; log weight. Fields and formula notes on the monitor template. Apply humane endpoints for ulceration or sharp weight loss.
- 07[Optional therapy] Randomize at a set volume (e.g. 100–150 mm³[1]); route and vehicle live in the protocol.
- 08[Endpoint] Weigh/process the s.c. tumor. Distant mets are not the intended readout—switch to orthotopic or experimental-mets protocols for metastasis questions.
- 09[QC] Fix strain, sex, age, passage, side, and inoculum; independent repeats. Troubleshoot before changing cell number.
Reagents / materials
| Item | Role | Conc. / dose |
|---|---|---|
| MDA-MB-231 cells (MC-h133) | Flank s.c. inoculum | 本 SOP 约 1–2×10⁶ / 100 μL；文献上限 2.5–5×10⁶[[2]] |
| Fetal bovine serum (FBS) | 10% complete medium | 常规培养优先 MC100 |
| Matrigel (typically 1:1) | Engraftment aid | 1:1 时约 50 μL/点，总体积 100 μL |
| Anesthetic / analgesic | Surgery & welfare | 按伦理批件与兽医 SOP |
| Test drug / vehicle (optional) | Treatment arm | 按药理方案 |
Readouts
s.c. tumor volume/weight, body weight, pathology; not a primary metastasis model.
References (PubMed)
- [1]PMID 34063098 — Evaluation of a Keratin 1 Targeting Peptide-Doxorubicin Conjugate in a Mouse Model of Triple-Negative Breast Cancer. Pharmaceutics (2021)
- [2]PMID 20422714 — Inhibition of human breast cancer xenograft growth by cruciferous vegetable constituent benzyl isothiocyanate. Mol Carcinog (2010)
- [3]PMID 33167919 — EP300 knockdown reduces cancer stem cell phenotype, tumor growth and metastasis in triple negative breast cancer. BMC Cancer (2020)
Disclaimer: RUO; IACUC required. Optimize by strain and pilot. Red tags mark weak or non-metastatic parental endpoints.