Seeding A549 cells into 96 wells plate.

Purpose: to seed approximately equal number of cells in each well of the 96 well plate and carryout the desire treatment.

• Cells were grown in 25 cm3 flask,remove media 
•  wash the cells with PBS or HBSS, add 2 ml of 1% trypsin for 90 second to make cells detached from the plastic and remove the trypsin 
•  monitor the cell rounding under the microscope for 5-7 minutes during that time tap the flask against your palm to expedite cell detachment.
• add 5-10 ml of media, thoroughly pipette cells up and down, transfer a 8 ml of the cell suspension to sterile tube for counting 
•  do a 1:10 dilution with vital stain as fallowing 50 ul cells : 400ul HBSS : 50ul trypan blue.
• use 10 ul of above cell dilution with vital stain for cell counting using hemocytometer 

cells/ml = (aver number of cells per large square )10000 * dilution factor 

total cells in tube = cell/ml * volume of cell suspension.

A 25000 cells per well were seeded in each well. 

Incubate the cells in 37 C ,5%CO2 for 48 hours to reach 90-100% confluent.