Ki-67 Intracellular Staining Protocol

Materials Needed

• Cell sample (PBMCs, splenocytes, tumor cells, or cultured cells)
• 70% ethanol (ice-cold, prepared fresh or stored at -20°C)
• 100% ethanol (for preparing 70% solution)
• PBS (phosphate-buffered saline)
• Fluorochrome-conjugated Ki-67 antibody (ethanol-compatible clone)
• Surface staining antibodies (optional, ethanol-compatible fluorophores)
• Blocking buffer (PBS + 2-10% FBS or normal serum)
• Wash buffer (PBS + 0.5-2% FBS)
• Staining buffer (PBS + 0.5% BSA or 2% FBS)
• 96-well V-bottom plate or flow cytometry tubes
• Ice bucket and -20°C freezer access
• Centrifuge
• Flow cytometer

Protocol

1. Pre-Experiment Preparation
   1. Prepare the plate layout or tubes required for testing, identifying all samples, controls (isotype/unstained), and serial dilution positions.
   2. Prepare antibody dilutions as required. Ensure fluorophores are protected from light.
   3. Place 70% Ethanol in a -20°C freezer at least 2 hours prior to use. Chill buffers on ice.
2. Cell Collection and Washing
   1. Collect cells for the flow assay and transfer to a conical tube.
   2. Wash cells once with 5-10 mL of Wash Buffer. Centrifuge at 500 x g for 5 minutes. Discard supernatant.
   3. Resuspend the cell pellet in Staining Buffer to the desired concentration. Aim for 0.5-1 × 10⁶ cells per sample.
   4. Add 100 μL of the cell suspension into each well of a 96-well V-bottom plate or to flow cytometry tubes.
   5. Spin down the cells (2400 rpm for 3 min) and carefully remove the supernatant by aspiration or flicking.
3. Surface Marker Staining (Optional)
    Important: Surface staining MUST be performed before ethanol fixation.
   1. Resuspend cells in Blocking buffer.
   2. Incubate for 10-15 minutes at 4°C to block Fc receptors.
   3. Add fluorochrome-conjugated surface antibodies at optimized concentrations.
      Note: Use ethanol-compatible fluorophores (PE, APC, PerCP/Cy5.5, Brilliant Violet series). Avoid FITC and some tandem dyes that may be affected by ethanol.
   4. Incubate for 20-30 minutes at 4°C in the dark.
   5. Wash cells twice with Wash buffer.
   6. Centrifuge at 300-400 × g for 5 minutes.
4. Fixation and Permeabilization (CRITICAL)
    Important: To prevent cell clumping, do not skip the vortexing step.
    For 96-well plates:
   1. Vortex the plate for 30 seconds. The pellet must be fully loosened into a 'slurry' before adding ethanol.
   2. While gently vortexing or shaking the plate, add 100 μL of pre-chilled (-20°C) 70% ethanol drop-wise into each well.
   3. Incubate the plate at -20°C for a minimum of 1 hour. (Cells can be stored here for up to 24h).
    For tubes:
   1. Resuspend cell pellet in 100 μL of PBS or Wash buffer.
   2. While vortexing gently, add 900 μL of ice-cold 70% ethanol dropwise to achieve a final concentration of ~70% ethanol. Critical: Add ethanol slowly while vortexing to prevent cell clumping.
   3. Incubate at -20°C for at least 30 minutes.
    Optional: Samples can be stored at -20°C for up to 1 week for batch processing.
    Note: Longer fixation (2-4 hours or overnight) often improves Ki-67 staining quality.
5. Staining and Acquisition
    For plates:
   1. Wash the plate 3 times with Wash buffer. (140 μL buffer, centrifuge 2400 rpm / 3 min). Ensure all ethanol is removed.
   2. Add 100 μL of Wash buffer into wells to rehydrate cells.
   3. Add 5 μL of the corresponding Ki-67 antibody (or dilution) to the wells. Mix gently.
   4. Incubate the plate at Room Temperature (RT) for 30 minutes, protected from light.
   5. Wash the plate 2 times with Wash buffer and centrifuge at 2400 rpm for 3 minutes.
   6. Resuspend cells in 60 μL of Staining buffer for flow cytometric analysis.
   7. Set the flow cytometer to acquire 40 μL of the sample volume.
    For tubes:
   1. Remove samples from -20°C and centrifuge at 400-500 × g for 5 minutes at 4°C. Important: Ethanol-fixed cells may pellet less tightly; use higher centrifugation speed.
   2. Carefully aspirate ethanol supernatant (pellet may be loose).
   3. Add 1-2 mL of Wash buffer to rehydrate cells. Incubate for 5 minutes at room temperature.
   4. Centrifuge at 400-500 × g for 5 minutes. Wash cells 1-2 additional times with Wash buffer.
   5. Resuspend cells in 100 μL of Blocking buffer (PBS + 5-10% FBS or normal serum). Incubate for 10-15 minutes at room temperature or 4°C.
   6. Add fluorochrome-conjugated Ki-67 antibody directly to the blocking buffer at optimized concentration.
   7. Mix gently and incubate for 45-90 minutes at room temperature in the dark. Use longer incubation times for potentially better signal.
   8. Wash cells 2-3 times with Wash buffer. Centrifuge at 400-500 × g for 5 minutes between washes.
   9. Resuspend cells in 200-500 μL of wash buffer or flow cytometry buffer.
   10. Optional: Add DNA dye (DAPI, PI, or 7-AAD) for cell cycle analysis if not using viability dye in same channel.
   11. Transfer to flow cytometry tubes if using plates.
   12. Keep samples on ice and protected from light until analysis.
   13. Acquire samples on flow cytometer within 4-6 hours for best results.

Expected Results

• Ki-67 Positive Cells: Actively proliferating cells will show bright Ki-67 staining, typically brighter than PFA-based methods.
• Ki-67 Negative Cells: Quiescent/resting cells (G0 phase) will show minimal to no Ki-67 expression with excellent separation from positive population.
• Typical Ranges:
  • Resting PBMCs: 1-5% Ki-67⁺
  • Activated T cells (48-72h post-stimulation): 40-80% Ki-67⁺
  • Tumor cell lines: 20-90% Ki-67⁺ depending on growth rate
  • Bone marrow: 10-30% Ki-67⁺ in healthy individuals

Tips and Best Practices

• Ethanol Preparation: Prepare 70% ethanol fresh from 100% ethanol and distilled water. Store at -20°C for immediate use. Do not use denatured ethanol.
• Vortexing is Critical: Always add ethanol dropwise while vortexing to prevent cell clumping. Clumps are difficult to break up after fixation.
• Temperature Control: Keep ethanol at -20°C until use. Cold ethanol provides better fixation and reduces cell damage.
• Batch Processing: The ability to store ethanol-fixed cells at -20°C for up to 1 week allows convenient batch processing of multiple samples.
• Fluorophore Compatibility:
  • Compatible: PE, APC, PerCP, PerCP-Cy5.5, PE-Cy7, APC-Cy7, Brilliant Violet series, Alexa Fluor series
  • Potentially Affected: FITC (may show reduced intensity), some PE-Texas Red tandems
  • Test First: Always validate new fluorophore combinations with ethanol fixation
• Antibody Titration: Titrate Ki-67 antibody with ethanol-fixed cells, as optimal concentration may differ from PFA protocols.
• DNA Staining: Ethanol fixation is ideal for combining Ki-67 with DNA dyes (DAPI, PI, 7-AAD) for comprehensive cell cycle analysis.
• Rehydration: Ensure complete rehydration before antibody staining. Insufficient rehydration can cause high background.
• Centrifugation Speed: Use slightly higher centrifugation speeds (400-500 × g) for ethanol-fixed cells as they pellet less efficiently.

Troubleshooting Guide

Problem	Possible Cause	Solution
Cell clumping after fixation	Ethanol added too quickly	Add ethanol dropwise while vortexing continuously; never add all at once
	Cells too concentrated	Dilute cells to ≤1 × 106/mL before adding ethanol
	Insufficient vortexing	Vortex at medium-high speed while adding ethanol; continue vortexing for 5–10 seconds after
Loose or no cell pellet	Normal for ethanol-fixed cells	Increase centrifugation speed to 400–500 × g; aspirate supernatant carefully
	Over-fixation	Reduce fixation time to 30–60 minutes; avoid storage >1 week
High background staining	Incomplete rehydration	Ensure thorough washing (3× minimum) after ethanol removal; include 5-min rehydration step
	Insufficient blocking	Increase blocking time to 15–20 minutes; use 10% serum
	Non-specific nuclear binding	Add DNase (10 µg/mL) during blocking step
Dim Ki-67 signal	Insufficient fixation time	Extend fixation to 2–4 hours or overnight at -20°C
	Antibody degradation	Use fresh antibodies; store properly as recommended by manufacturer
	Wrong antibody clone	Try alternative Ki-67 clone validated for ethanol fixation
Loss of surface markers	Ethanol damages epitopes	Perform surface staining before fixation; use ethanol-compatible fluorophores
	Fluorophore incompatibility	Switch to PE, APC, or Brilliant Violet conjugates; avoid FITC
High CV (coefficient of variation)	Cell clumps present	Filter samples through 35–40 µm strainer before acquisition
	Incomplete mixing during staining	Vortex gently after adding antibody; mix periodically during incubation
	Aggregated antibody	Centrifuge antibody at 10,000 × g for 5 min before use