Cell Maintenance Protocol

Lab Protocol
Human adult low Calcium high Temperature (HaCaT) keratinocyte cell
line
Media: DMEM, 10% heat-inactivated FBS, 1% PenStrep, 1% Kanamycin
Sulfate, 0.8% Sodium bicarbonate
Wash: DPBS 1X, HBSS 1X
Dislodged by 0.25% Trypsin
1. Safety measures
a. Never work unattended
b. Lab coat, gloves, and goggle at all times
c. Secure lab door when entering and exiting lab
2. Close Window of the Biosafety Cabinet
a. Turn on UV Light for a minute
b. Turn on Vacuum and Power
c. Turn off UV light
3. Open Window of Biosafety Cabinet
a. Use Ethanol and Lab Soap and wipe down area
b. Spray down your hands with 70% ethanol whenever you
enter or exit the hood
c. Practice aseptic technique and avoid touching any other
surfaces other than the ones you are working one
d.
Warm the media and wash in the water bath set at 37C. Do not ever
put cold media or wash onto cells.
Take cell Flask out of incubator
Take out PBS, Medium, and .
4. Sterilize all containers before putting into Cabinet. Bring
everything you need into the hood before taking out the flask.
5. Visualize your cell line under the microscope any peculiarities.
a. Using the vacuum suck the medium out of flask.
b. Be sure to not touch the side with the cells
6. Using the electric pipet, suction PBS and place into flask
a. Be sure to not touch the side with the cells
b. Rock the flask back and forth to wash the entire surface.
c. Vacuum the PBS
7. Use the electric Pipet to put 3-4 ml of trypsin put . to To
dislodge the cellsbreak the enzymes of thefrom the side of the
wallbase of the flask. It is okay to touch the side of the wall while
doing this.
a. Incubate the flask for 5-7minutes @ 37C.
b. Tap the sides post-incubationGive the flask a few shakes.

8. Use electric pipet to put 6-7 ml of some medium into the fFlask in
order to neutralize the trypsin.
9. Use the electric pipet to suction up the cells, , trypsin and
the medium. Dont forget to wash the sides down several times
to dislodge any remaining cells.
a. Place into a 15ml conical test tube.
10.
Place tube test tube into centrifuge
a. Be sure to balance the Centrifuge
b. Set to 1100RPM
c. Set to 5 minutes
d. Room Temperature
11.
Take new flask
a. Label appropriately
i. Date
ii. Hacat and Passage
iii. Initial
b. Place 25ml Medium into the Flask
c. Place the new flask into the incubator in order to condition
the medium to 37C and 5%CO2 for 5-7 min
12.
Take out a dual chamber plate (24- well transwell tissue
culture plate)
a. Place top (apical) chambers (the top chamber is called an
insert) to one side.
b. Fill the open chambers on the bottom (basolateral) with
600ul of medium
13.
Once centrifuge is done, take out tube
a. Suction out all the fluid but leave behind the ppeallet of
cells
b. Flick tube to break up peallet.
c. Resuspend the pellet with 10ml of medium and pipet up
and down against the walls of the tube in order to
homogenize the entire suspension. You will call this your
cell suspension**** and you will use this to seed your
cells and reculture them.
14.
Set a 10-100ul micropipette to 50ul and aseptically pipette
out a solution,
a. When transferring bio liquids in and out of their containers
make sure the top and the bottom of the cap never comes
in contact with any surfaces including your hands.
15.
Transfer 50ul of cell solution to a sterile 0.5ml Eppendorf
tube
16.
Pipet equal parts of trypan blue. Be sure to pipet from the
surface of the dye to avoid picking up any residue from the
bottom. Transfer 50ul of trypan blue to the Eppendorf tube and
resuspend

17.
Resuspend the cells and transfer it over to a
hemacytometer
18.
Carefully and lightly anchor the cover slip and pipet the
suspension into the slit to drive capillary action
19.
Use a light microscope to visualize cells and count
accordingly
20.
Splitting and seeding cells
a. After counting with the hemacytometer, you are essentially
adjusting your original cell solution to one that is needed
for experiments or culturing. You can figure out how much
of the solution you need to reach a desired concentration
in a volume of media

Counting on the Hemocytometer: Trypan Blue Exclusion Test

-After obtaining counts determine total number of cells

present
Determine Total Number of Cells Present

* Total cells counted = Average of counts

**Dilution Factor = 2 (1:1 mixture of cells and Trypan Blue)
Drawing from your own experience from counting: After youve
counted i.e. 300 cells in total, (300 x dilution factor (2) / # of squares
(4) x 10,000 cells/ml) you get 1.5 x 10^6 cells/ml in 10ml of
suspension which is a grand total of 15 million cells. This is the
concentration of cells that you have.
Those top chamber wells (inserts) can hold up to 400ul of liquid. You
want to put just the right amount of cells so that the cells arent too
sparse or too crowded.
The action of putting a specific concentration of cells into a well is
called seeding. You will seed 1.5 x 10^5 cells/ml in a volume of 200ul
in each well. This is the desired concentration you want to seed at.
So how many wells will you be making? Usually 12 but you should
overestimate to account for instrument errors. So lets say 15 wells, Vtotal would 15 x 200ul = 3000ul or 3ml (450,000 cells in total).
This is the total volume of cells you want.

CdesiredV-totaldesired=ChaveVx
Whats the desired concentration you want to seed at?
Whats the total volume of cells you want?
What is the concentration of cells that you have?
Find Vx
Vx is how much you will pipet out from your 10ml cell suspension in
order to get 450,000 cells in total. You will get that exact number of
cells in a small volume (usually under 1ml). So dilute that volume up
to a total volume of 3ml which is your desired volume of cells at your
desired concentration.
150,000 cells/ml x 3ml = 1.5 x 10^6 cells/ml x Vx
Vx = 300ul of cell suspension
3ml 300ul = 2.7ml of medium
21.
So in a new 15ml conical tube pipet in 2.7ml of medium
and 300ul of cell suspension to get 150,000 cells/ml in 3ml of
medium.

22.
Homogenize thoroughly and pipet 200ul of that new cell
suspension youve made into each insert. Remember that earlier
you filled the bottom chamber with 600ul of medium.
23.
Now that youve seeded your experiment plate. You will
culture your cells back into a new flask. Go back to your old cell
suspension and calculate the amount of cells you want to seed
back into the big flask.
24.
Using the same concept as above, I usually put 200,000300,000 cells in total into the big flask (which has 25ml of
medium) that youve prepared beforehand.
25.
In this case: ignore V-total, I just want 200,000-300,000
cells in total into the big flask. So 250,000 cells = 1.5 x 10^6
cells/ml x Vx
26.
Vx= 167ul, so I just simply pipet that amount from the old
cell suspension into the big flask and you have successfully
recultured/splitted your cells.
27.
You may dispose the old and new cell suspension once
everything is seeded
28.
Place the experiment plate and flask into the incubator @
37C with 5%CO2.
29.
Feeding (changing/replenishing with new medium)
a. Cells should be fed according to its density, it is important
that cells have enough food to be metabolically active.
b. Cell waste can be toxic to an overcrowded population,
changing media will eliminate that.
c. Visualize your cells under the microscope and note the
density, morphology and any peculiarities
d. Vacuum out the old medium and replenish with 25ml of
fresh medium.
e. Incubate your cells at 37C/5%CO2. Cell population
doubling time is 24hrs.