Basic Cell Plating and Cell Culture Maintenance Protocol

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About Cell Plating

In order to culture cells for standardization of an experiment such as drug dosage, cytotoxicity, transfection or cell migration assays etc., a fairly good number of cells are required.

In such a case cells are cultured on plates having wells. There are 6/ 24/ 96 well plates used routinely for such experiments.

Why Cell Plating Required?

Wide applications of eukaryotic cell culture systems and need of compiling maximum, reliable, reproducible data has given rise to the necessity of miniaturization of this system that led to the development of cell based assays.

This has become possible by cultivation of cells in small vessels such as 96 well plates or 6 well plates.

Seeding cells in dishes or multiwell plates is called as cell plating.

This gives an opportunity to increase number of samples to be assayed with high reproducibility.

The ability to gather more than one set of data from the same sample (i.e., multiplexing) can contribute to saving time and effort during screening.

Multiplexing can provide internal normalization controls to confirm the results of other assay methods and eliminate the need to repeat work.

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Adopted from BioRender

Cell Plating Requirements


1. Growth medium

2. Trypsin (0.25%) and1 mM EDTA in PBS

3. 96, 24, 6 well tissue culture plates

4. Pipette tips

5. Reagent reservoir

6. Multichannel pipette

7. Tubes, 15 ml, 50 ml

8. Discard Beaker

Non Sterile:

9. 70% IPA

10. Cotton/tissue paper

Cell Plating Procedure

1. View cultures using an inverted microscope to assess the degree of confluency and confirm the absence of bacterial and fungal contaminants.

2. Remove spent medium. If the cells are in suspension, centrifuge it in 15 or 50 ml sterile tube at 1300 rpm for 7-10 min and proceed to step 6.

3. Wash the cell monolayer with PBS without Ca+2 Mg+2 using a volume equivalent to half the volume of culture medium. Repeat this wash step if the cells are known to adhere strongly.

4. Add enough trypsin/EDTA at 37oC to cover the cell layer (~3mL in T75, ~2.0mL in T25).

5. Incubate for 2 minutes at 37oC in CO2 incubator. Tap occasionally to verify that the cells are releasing. Check in microscope to visualize detachment of cells.

6. Remove trypsin-EDTA. Add fresh 2 ml of medium and rinse cell layer two or three times to dissociate cells and to dislodge any remaining adherent cells.

7. Remove 100-200μl aliquote and perform a cell count (Experiment 1.3- cell counting).

8. Make cell dilution in a reagent reservoir to plate appropriate cell number according to plate used. For instance for 96 well plate 10^4 cells/ well are plated.

9. By using a multichannel pipette, plate cells in multiwell plates.

10. Incubate at 37°C in 95% air for 24-hours.

Cell Plating Citations:


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