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Handbook of Cell Culture Plate Tips & Tricks
This guide covers essential tips from selection and setup to operation and analysis.
Section 1: Pre - Experiment Preparation & Selection
Choose the Right Plate:
Golden Rule: Match the plate to your detection method. Use clear plates for Absorbance /ABS, black walled & clear bottom plates for Fluorescence /FL, and white walled & clear bottom plates for Luminescence /LUM. Using the wrong plate causes signal crosstalk and poor data.
Adherent Cells: Always select a Tissue Culture Treated surface.
Suspension Cells & Spheroids: Use an Ultra Low Attachment surface.
Imaging: For high quality microscopy, prioritize glass bottom plates.
Labeling Smartly:
Never label the lid! Lids can easily be swapped or misplaced. Always label the side of the plate.
Use a lab grade, alcohol resistant, and cryogenic proof marker. Avoid Sharpies as they can be wiped away by ethanol.
Pre - draw a grid on the lid with a marker for easy visual navigation to specific wells.
Warm Everything Up:
Allow plates especially TC treated ones and media to warm to room temperature inside the biosafety cabinet before seeding cells. Cold surfaces and media drastically reduce cell attachment efficiency.
Section 2: Seeding & Culture Techniques
Minimize the Edge Effect:
The outer perimeter wells of a 96 well plate evaporate faster, altering media concentration and causing uneven cell growth this is the "edge effect."
Solutions:
Best Practice: Fill the outer perimeter wells with PBS or sterile water to humidify the plate and minimize evaporation in the inner experimental wells.
Good Practice: Do not use the outer wells for experiments; use them for blanks or leave them empty.
Use specially designed low evaporation lids or sealing films.
Cell Seeding Tips:
Mix Thoroughly: Gently but thoroughly resuspend the cell suspension immediately before seeding to ensure a consistent number of cells per well. This is critical for reproducibility!
Figure 8 Swirling: After seeding, gently swirl the plate in a figure 8 motion on the bench to distribute cells evenly across the well bottom and prevent central aggregation.
Avoid Bubbles: Keep pipette tips away from the liquid surface when dispensing to avoid creating bubbles. Bubbles disrupt microscopy and plate reader readings. Remove them with a sterile needle or by brief centrifugation, e.g., 1000 rpm for 1 minute.
Media Change Techniques:
When aspirating with a multichannel pipette or vacuum pump, hold the tip vertically and avoid touching the bottom of the well to prevent damaging or dislodging cells.
Use a gentle vacuum setting or a manual multichannel pipette for more control to avoid accidentally aspirating cells.
Section 3: Detection & Analysis Tips
Pre - Readout Preparation:
Maintain Sterility: If cells need to be cultured after detection, ensure all subsequent steps are performed under sterile conditions.
Wipe the Bottom: Before placing the plate in a microplate reader, gently wipe the bottom with a lint free tissue moistened with ethanol or water to remove fingerprints, dust, and condensation. This is crucial for accurate optical readings.
Controls are King:
Always include the necessary control wells:
Blank Control: Media only, no cells. Used to baseline the plate reader.
Negative Control: Untreated cells.
Positive Control: Cells treated with a compound of known effect.
Section 4: Other Pro Tips
Prevent Cross Contamination:
When using a multichannel pipette, check that all tips are dispensing equally and that no liquid is clinging to the outside of tips.
Use dedicated pipette tip boxes and reservoirs for different experimental groups to avoid reagent carryover.

Sealing and Storage:
For longer term cultures more then 3 days or shipping, use a gas permeable seal to allow for gas exchange while preventing contamination and evaporation.
For sample storage or to terminate an experiment, use an aluminum seal for a complete, airtight closure.

Cost Saving Hack:
For pilot experiments or assays where top precision is not critical, plan multiple experimental conditions on the same plate to maximize well usage. Just be sure to include appropriate controls and account for positional effects.
Summary: Golden Rules
Plan First: Draw a plate map before you start. Know what goes in every well.
Mix is Key: Inconsistent cell & reagent seeding is the number one cause of failed experiments.
Pamper the Edge: Save your data by filling the perimeter wells with PBS.
Keep it Clean: Wipe the plate bottom before reading treat it like a camera lens.
Controls Rule: Without proper controls, your results are meaningless.
Mastering these tips will significantly enhance your experimental efficiency, reproducibility, and success rate!