Migration 24well Transwell:

Migration Assay on 24 well plates

Used wells: Transwell from Costar with 6.5mm diameter and 3.0 µm pore size

Discription:

The Migration chamber has 2 chambers separated with a membrane: The lower chamber contains the chemoattractant, the upper chamber contains the cell suspension

Procedure:

• Add 700 µl of medium containing the desired concentration of chemoattractant into the lower chamber
• Insert the membrane
• Add 100 µl of cell suspension (c: 107/ml, ie 106 /well)
• Incubate 2-4 hours (37C, 5% CO2)
• • Assess the migration after 2, 3, 4 hours under and inversion microscope, looking at the cell number on the bottom (migr. Cells) and on the membrane (not migrated cells), in order to find the best time to stop the migration assay
• Take out the membrane and add 140µl MTS-mix per well (see MTS-Assay)
• Incubate 1-4 hours (depending on visible color change) in 37C, 5% CO2
• Read absorption on plate reader (490 nm)

Negative controls:

- cells migrated toward medium containing no chemoattractant

- Medium alone (Blank for MTS assay)

Positive controls:

- cells migrated toward know chemoattractant (LPS, fMLP, Eotaxin 100ng/ml)

- 106 cells as pos control for MTS assay

MTS Mix:

Mix 5 µl of 10 x PMS with 1ml of MTS

Add 20 µl of PMS/MTS-Mix to 100µl Cell Susp (ie: 140 µl MTS-mix / migration well)

At the end of the migration assays, cleanse the filter side of the upper chamber with a cotton swab and stain the filter for one hour with crystal violet (Sigma) in 2% ethanol and then rinse in water.
7) Gently cut the filter from the chamber and count the cells that have migrated through the filter pores from the underside of the filter.
8) Count the number of cells that migrated across the filters in 4 high-power fields per insert, and average values afterwards. For each migration condition, three identical replicates should be performed.

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    细胞迁移实验如果用chamber做的话，无论用哪家的产品原理都是一样的，所做的改进只是结果更加精确，主要是计算迁移细胞的方法上的进化：

    1 用结晶紫染色后在显微镜下数细胞数目；

    2 将染过细胞的结晶紫或其他染料，比如MTS Mix，冲洗下来后放到酶标仪上读数，减少误差

    3 用荧光染料染色后，测量发光效果。

   最省钱的方法当然是scratch。