Skip to Main

Colonic/Small Intestinal LPL — Myeloid Macrophage Identification Panel

  • Tissue: Colon/small intestine
  • Species: Mouse
  • Instrument: Cytek Aurora or BD Analyzer
  • Colors: 4 (screening panel)
  • Application: Myeloid/macrophage identification

Antibody Panel

#TargetCloneFluorochromeLaser/detectorRole/cell population
1 Visability dye -- Ghost dye 710 Red (R4) → ~710 nm Dead cell exclusion. Ghost dye = live cells
1 CD45 30-F11 BUV395 UV 355 nm → ~395 nm Pan-leukocyte marker. Gate CD45⁺ leukocytes from stromal cells.
2 CD64 (FcγRI) X54-5/7.1 StarBright Blue 700 Blue (B9) → ~700 nm Macrophage-specific Fc receptor. CD64⁺ distinguishes macrophages from monocytes.
3 CD11b (Mac-1) M1/70 FITC Blue 488 nm → ~525 nm Myeloid cell marker. CD64⁺ CD11b⁺ = lamina propria macrophages.

Gating Strategy

StepX-axisY-axisGatePopulation & Criteria
1 FSC-W SSC-W Scatter Gate Exclude debris and aggregates. Intact cell cluster. Gate: ~99.9% of events.
2 SSC-A SSC-H Single Cells 1 Doublet exclusion on diagonal (SSC-A ≈ SSC-H). ~90.9% of scatter gate.
3 Ghost 710 (AF700) CD45 (BUV395) Viable CD45+ Ghost Dye⁻ CD45⁺ = live leukocytes. Upper-left quadrant. ~45% of single cells (78,563 events).
4 CD64 (SBB700) CD11b (FITC) Macrophages CD64⁺ CD11b⁺ double-positive = lamina propria macrophages. ~19.1% of viable CD45⁺.

Gating Hierarchy

  1. Scatter (FSC-W/SSC-W)
  2. Singlets (SSC-A/SSC-H)
  3. Viable CD45⁺ (Ghost 710⁻ CD45⁺)
  4. Macrophages (CD64⁺ CD11b⁺)

See the graphic below for representative gating plots.

Sequential Gating Strategy — LPL Macrophage Identification

Small Intestinal Lamina Propria: Representative density plots show the four-step myeloid macrophage gating workflow.

Figure: Representative flow cytometry density plots from small intestinal lamina propria. Each plot shows the gate applied at that step and the percentage of the parent population captured. Gate 4 (CD64⁺ CD11b⁺) identifies tissue macrophages and represents approximately 19% of viable CD45⁺ leukocytes.

Intestinal Lamina Propria (LPL) Isolation Protocol

Tissue-specific note:

  • Small intestinal LPL:  HBSS + 3% FCS + 20 mM EDTA for the epithelial stripping step. Remove Peyer's patches before digestion.
  • Colonic LPL:  substitute 10 mM EDTA + 1 mM DTT — DTT disrupts the colonic mucus layer for better LP cell access.
  • Both protocols use the same digestion and Percoll gradient steps below.

Materials

  • HBSS, 37°C, filter-sterile (no phenol red, no Ca/Mg; + NaHCO₃ 4.17 mM) + 3% FCS + 20 mM EDTA
  • Colonic LPL variation: substitute 20 mM EDTA + 1 mM DTT (DTT loosens mucus)
  • Digestion solution: RPMI +4.17 mM NaHCO₃ +5% FCS +Collagenase IV (125 µL/50 mL) +DNase I 0.1 mg/mL (250 µL/50 mL) +Dispase II (250 µL/50 mL)
  • 100 µm and 70 µm cell strainers
  • 40% and 80% Percoll (room temperature)
  • Gavage needle and 10 mL syringe
  • Sterile 50 mL and 15 mL conical tubes
  • Sterile bottles and Erlenmeyer flasks
  • Steritop 0.22 µm filters
  • Large volume ice-cold PBS

Procedure

  1. Harvest intestinal tissue; place in ice-cold PBS +3% FBS in a Petri dish on ice.
  2. Flush the lumen with 10 mL ice-cold PBS via gavage needle to remove luminal contents.
  3. Remove Peyer's patches (small intestine). Cut intestine open lengthwise with scissors.
  4. Cut into ~1-inch pieces. Transfer to 50 mL conical with 10 mL ice-cold 3% PBS.
  5. Wash tissue 2× with 10 mL ice-cold 3% PBS: vortex 5 min (~10–12 inversions), pour off supernatant, repeat.
  6. Epithelial stripping: add 10 mL HBSS + 3% FCS + EDTA (see tissue note above). Rotate at 250 RPM, 37°C, 15 min. IEC collection: if collecting intraepithelial cells (IECs) for flow, incubate on ice with gentle vortexing every 5 min instead.
  7. Vortex 2 min. Pour off supernatant (contains IELs). Repeat step 6 once more.
  8. Enzymatic digestion: add tissue pieces to 10 mL RPMI digestion solution (Collagenase IV +DNase I +Dispase II). Incubate 37°C, 250 RPM, 45 min. Tip: do not over-digest; 45 min is the maximum. CD64 surface expression can be reduced by excess collagenase.
  9. Vortex 2 min. Pass supernatant through 100 µm → 70 µm cell strainers into a 50 mL conical.
  10. Second digestion of remaining tissue: 30 min, 37°C, 250 RPM. Pool supernatants from both digestions.
  11. Centrifuge pooled supernatant to pellet cells. Discard supernatant.
  12. Percoll gradient: resuspend pellet in 10 mL room-temperature 40% Percoll. Carefully underlay with 3.5 mL 80% Percoll in a 15 mL conical.
  13. Centrifuge: 800 × g, 20 min, no brake, room temperature.
  14. Collect buffy coat from the 40%/80% interface. Wash 1–2× with ice-cold media.
  15. Count cells. Proceed to antibody staining per the UTSW General Flow Cytometry Staining Protocol.

Tips for optimal macrophage recovery

  • Macrophages sediment below the interface — also collect just above the 80% Percoll layer when transferring.
  • Collagenase IV lot variation affects digestion efficiency and CD64 surface expression. Titrate each new lot.
  • If CD64 signal is weak: reduce Collagenase IV concentration or add a brief Fc block (anti-CD64 clone 93) post-isolation.
  • Add CountBright or similar counting beads before acquisition for absolute cell counts.
  • For sorting macrophages: substitute the Sorting Staining & Sample Prep protocol and skip fixation.
Back-to top