Lab instructions plating 3T3 fibroblasts on a 24 well plate

Lab instructions: plating 3T3 fibroblasts on a 24 well plate (4x6) using 2 different substrates. and one without using a substrate. 1x10^4 cells in each plate.

trying to test how cell proliferaton is affected by the use of 2 different subsrates, comparing them to a control (one witout substrate used).

Media is already made : 90 mL DMEM (stored at 4 °C) and 10 mL FBS (fetal bovine serum, stored at -20 °C, thaw in water bath)

--> Please list the steps on How to plate cells on extracellular substrates after cells are subcultured.

Solution

Answer:

steps on How to plate cells on extracellular substrates after cells are subcultured.

1- Calculate the required volume of media:

There are 3 experimental set ( 2 set for substrates and one for control) and each set contais three wells i.e. in triplicate. so total 9 wells are required out of 24 wells in a plate. In a 24 well plate, each well has 1ml working volume. so we require 9 ml media. But to avoid pipetting error we have to take total of 10 ml of thawed DMEM media in a separate sterile 15ml tube.

2- Add FBS to the media:

We have to take 10% of FBS and add to the media and mix well by vortex

* All the work must be done in a bio-safety cabinet.

3- Calculate the number of 3T3 fibroblast sub-cultured cells required

For each well, 1x10^4 cells require.

So for 10 wells we require 1x10^5 cells total.

Then count the cells by cell counter and add to the complete media (FBS+Media) and vortex well.

4- Then mark the plate according to the experimental set

5- Then for each set, extract the cell containing media and and add substrate according to the marking on a plate.

and remember there is no substrate to be added in a control set.

6- cover the plate and incubate in a cell incubator