Inhibited Migration to the Lower Chamber
This issue may result from an insufficient chemotactic gradient or the absence of effective chemoattractants in the lower chamber. To address this, consider serum starvation of the cells for 12–24 hours prior to the assay to synchronize the cell cycle. Ensure appropriate negative and positive controls are included. You may also increase the concentration of serum or other chemotactic agents in the lower chamber. Additionally, excessive cell passaging may compromise cell viability and migration ability; therefore, low-passage cells are recommended. Another critical factor is the pore size of the Transwell insert—pores that are too small may impede cell migration, so using inserts with larger pore sizes may help. For invasion assays, overly thick Matrigel layers can also obstruct migration; consider reducing the volume and optimizing incubation time during preliminary experiments. Lastly, be cautious of air bubbles beneath the membrane insert, as they can interfere with cell migration—take care to eliminate bubbles during setup.
Overmigration or Undermigration of Cells
It is recommended to perform preliminary optimization experiments using the control cell line to determine the appropriate cell seeding density and incubation time. Poor cell condition at the time of seeding, inaccurate cell counting, or incorrect volume calculations may all affect migration. If cell viability is low during counting, the experiment should be postponed. Ensure thorough resuspension of cells before counting and again prior to seeding to maintain uniform distribution.
Uneven Cell Distribution Post-Staining
To ensure uniform distribution, make sure the inserts are placed on a level surface. Mix the cell suspension thoroughly before seeding. After seeding, allow the inserts to sit undisturbed in the biosafety cabinet for a short period before transferring them to the incubator. Handle inserts gently during transfer to avoid tilting or vibration, and close the incubator door carefully to prevent shaking. If severe cell aggregation is observed, increase the number of washing steps or change the culture medium to remove clumps and aggregates.
Inconsistent Experimental Reproducibility
First, evaluate whether inaccuracies in cell counting or failure to mix the cell suspension prior to seeding could be contributing factors. It is advisable to mix the suspension each time before adding cells to an insert. Standardizing the experimental protocol and using reagents and consumables from the same batch can also improve reproducibility.
Lack of Distinct Differences Between Experimental and Control Groups
Several factors should be considered: ensure that the membrane pore size is appropriate for the cell type; avoid using overly passaged cells, which may have reduced migratory potential; confirm that the chemoattractant gradient was correctly established during setup. Including a positive control in each treatment group can help identify procedural errors. Prior to imaging, clean the upper surface of the insert thoroughly to remove residual cells or stains that may interfere with observation.
Ambiguous Visualization of Migrated or Invaded Cells
This may be due to high background staining. Use clean reagents and handle the insert membrane carefully when wiping to remove background—multiple gentle wipes may be necessary. As the insert membrane is porous, take care to differentiate cells from membrane pores during imaging. If no elongated or spread morphology is observed, the incubation time may have been too short; extending the incubation period may improve visualization of invasive or migratory cells.
Product Overview
|
Product Name |
Specification |
Cat No. |
|
1 roll |
36125ES03 |
|
|
20 T/50 T/100 T |
40302ES20/50/60 |
|
|
20 T/50 T/100 T |
40305ES20/50/60 |
|
|
10 mL/100 mL |
40202ES80/92 |
|
|
1 mL/5×1 mL/50×1 mL/100×1 mL |
11184ES03/08/50/60 |
|
|
1 mL/5×1 mL |
11188ES03/08 |
|
|
1 mL/5×1 mL |
11189ES03/08 |
|
|
1 mL/5×1 mL |
11190ES03/08 |
|
|
1 mL/5×1 mL |
11171ES03/08 |
|
|
20 T/200 T |
11175ES20/70 |