Protocols_CellCulture_Culturing HEK293T cells and its variants - BackmanLab/BackmanLab.github.io GitHub Wiki

Culturing HEK293T cells and its variants

Preparation

  1. Before starting the procedure, ensure that all reagents are at 37$℃$ by keeping them in the hot water bath for 20 to 30 mins. PBS can be stored and used at room temperature.

  2. Make sure the inside of the hood is entirely sprayed and wiped with 70% Ethanol/Isopropanol. Any item that goes inside the hood must be sprayed and wiped with 70% Ethanol with no exception.

NOTE: Reagents must not be heated beyond 37$℃$ and over 30 minutes as these will degrade the components that are necessary for optimal cell growth.

  1. Check the confluency of the cells under the Microscope. Split only if they are around 70% - 80% confluent. (20% black space on dish)

Thawing cells

Rapidly thaw the cells by placing them at 37°C in a water bath with gentle agitation for 1--2 minutes.

NOTE: Freezing Medium may be yellow immediately after thawing. This does not affect cell viability if these instructions are followed.

  1. Decontaminate the vial by wiping it with 70% ethanol before opening in a class II biological safety cabinet.

  2. Slowly transfer the vial contents into 10ml of Growth Medium in a sterile 15ml conical tube.

  3. Centrifuge the cells at 500 x g for 5 minutes at 18°C.

  4. Aspirate the supernatant and resuspend the cell pellet in 12ml of 37°C prewarmed Growth Medium.

  5. Transfer resuspended cells to a 100 mm dish. Store in 37C 5%CO2 incubator. Make sure incubator has water in the pan on the bottom to maintain optimum humidity.

Washing

  1. Aspirate the old media.

  2. Perform a wash with 1 ml PBS, swirl gently and discard it into the disposal bottle.

Dissociation/Trypsinization

  1. Add 1 ml of Trypsin or 2ml TrypLE express into the side of the flask. Gently rock the flask to ensure the entire surface is covered with it.

  2. Place the flask in the primary incubator for approximately 1 to 2 minutes to ensure quick trypsinization.

  3. Check if all cells have dissociated well by viewing the flask under the microscope. If some are still attached, gently tap the flask onto the desk a couple of times and then slightly tap it with fingers at the sides of the flask giving it a little bit of a shake.

Trypsin inactivation and removal

  1. In the hood, add x ml of respective media to make upto 10ml cell suspension (Trypsin to Media ratio must be 1:3) to the flask and wash the surface of the flask well by pipetting the suspension against it twice. Transfer this cell suspension into a 15 ml centrifuge tube and label it respectively.

NOTE : Check if the centrifuge is clean and empty prior to keeping the cell tube in it.

Cell counting (ask Che how they do it in IGB)

This protocol is for using a hematocytometer. Some labs use an automatic cell counter.

  1. Take 15 $\text{μl}$ of suspended cells and transfer to the 0.5 ml tube and then add 15 $\text{μl}$ of Trypan Blue and mix these well by re-pipetting. The cell to stain ratio must be 1:1.

  2. Add 10 $\text{μl}$ of the stained cells to the hematocytometer (cell counter, ask Che where it is in IGB).

  3. Determine the cell count. See protocol here: https://bitesizebio.com/13687/cell-counting-with-a-hemocytometer-easy-as-1-2-3/

Seeding/Plating cells

  1. According to the cell line, determine what would be the optimal

    seeding density and calculate the volume of cells needed to obtain that density.

NOTE 6: Do not seed at less than 20%-25% density as the cells will not be able to grow because of sparsity.

  1. Put the respective volume of media in a new flask.

  2. Add the calculated volume of cells into the new flask. Gently mix

    the cells in the media by grazing the flask back and forth on the work area surface to ensure even distribution.

  3. View the flask under the microscope to ensure even distribution of

    cells.

  4. Place the flask carefully in the primary incubator. Make sure to not

    rock it too much so as to avoid disturbing the even distribution of the cells.

  5. Allow cells to grow for respective no. of days in the incubator at

    37$℃$ and 5% CO2.

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