Sorry, wrong spelling, I use 2 mM of CaCl2 in one well, or 2mM of EDTA in another well that has the same transfected cells as control. My cells aggregate in the EDTA well too.

Thank you for telling me!

Check the recipe of your HCMF buffer. I have seen several different buffers called HCMF. Some of them contain calcium, some don't. If yours does, then 2 mM EDTA may be insufficient to bind it all.

Thank you! My HCMF buffer does not have calcium in it, but my tripsin does so I might try to take out the calcium in the tripsin and try again.

I'm writing here my whole protocol, I think it might be easier to check if something is not right.

- BUFFERS:

o Tripsin +CaCl2 :100 Ml of tripsin +200 µL of CaCl2 1M. Filtered.

o 1M CaCl2: M:110.98 g/mol. 22.196 g in 200 mL water.

o HCMF buffer: 137 mM NaCl, 5.4 mM KCl, 0.63 mM, Na2HPO4, 5.5 mM glucose, and 10 mM Hepes, pH 7.4. Filtered.

 NaCl: 58.44 g/mol: 8 g.

 KCl: 74.55 g/mol: 0.4 g.

 Na2HPO4: 141.96 g/mol: 0.089 g.

 Glucose: 180.16 g/mol: 0.99 g.

 HEPES: 238.3 g/mol: 2.38 g.

o 1% agar: 0.5 g in 50 mL H2O or PBS. ( CONSERVED IN THE FRIDGE)

o EDTA: 292.24 g/mol.100 mL at 100 mM, 2.92 g in 100 mL, pH adjusted to 8, autoclaved.

- Cells were trypsinized in the presence of 2 mM CaCl2, pelleted, and resuspended in HCMF buffer at a concentration of 2.5 × 105 cells/ml.

- Cells were divided in two tubes, one had added 2 Mm EDTA and the other 2 mM CaCl2.

- For each condition, 0.5 ml of the cell suspension was added to 1% agar-coated wells with coverslips (70 µL) in a 24-well plate.

- Cells were shaken for overnight at 80 rpm at 37°C, and images of random fields were collected on a microscope using a 10× objective.

Thank you again for the help!