Lab Task

Cell culture

Submitted by
Muhammad Fozan
Department of Bio-medical sciences
Pak-Austria Fachhochschule
Institute of Applied sciences and Technology, Haripur

Class In-charge
Dr. Imran Khan

Date
December 16, 2022
Antibiotic stock solution
Antibiotics are routinely used in cell culture, in order to prevent contamination from bacteria,
maintain aseptic condition for maximal cell growth. Here, in our experiment we used Ampicillin
(Penicillin) as antibiotic in DMEM media for growth of Bone marrow derived stem cell.

Preparation of 1% Ampicillin stock

 Take 0.1g Ampicillin powdered into sterile conical flask.

 Add 8 ml of Ro water into flask, shake gently in order to avoid bubble formation.
(*Vortex at slow speed)
 Dissolve as possible precipitate, it would take about 30 min for complete dissolution.
 Fill up-to 10 ml mark with Ro water.
 Now, filter the antibiotic solution with syringe filter (0.22µm). Don’t autoclave solution.
 Then, convert the filtered solution into 2 ml aliquot for further use. ( store at -200C)

Cell culture media

Cell culture media is optimal environment for cell growth, which is composed of amino acid,
protein, vitamin, and other growth factor which promote the cell growth. Here, we use
DMEM media, which support the adhesion of cell. Initially, we isolate the stem cell from
bone marrow of femur Via PBS, and added it to media.

Calculation for Fetal Bovine Serum

According to literature we use 10% FBS into media, which support cell growth as well as
proper nutrition.

Here, we are making calculation for 25 ml of total volume;

10% in 25 ml ----------------------------------------------10/100*25=2.5 ml of FBS

Take 2.5 ml of FBS into 50 ml conical flask.

Calculation for Antibiotics

 According to literature, we have to use 1% antibiotic for prevention of bacterial
contamination.

As mentioned above, we make 1% antibiotic stock solution. For 25 ml volume, we have

following calculation.

1% antibiotic in 25ml-------------------------------------------------1/100*25=0.25ml

Take 250 µl of antibiotic from stock solution into media

Preparation of media

 Prepare the culture hood priorly to use. Clean the hood with 70% ethanol.
 Transfer every item into hood after cleaning with 70% ethanol.
 Take 50 ml conical flask and transfer 20 ml of DMEM with disposable pasture
pipette.
 Add 2.5 ml of FBS and 0.25 ml of Antibiotic into flask. Fill the remaining volume
up-to 25 ml with DMEM.
 Gently, mix the constitute into media via titration or shaking.
 Seal each tube with Parafilm in order to protect from contamination.

PBS formation

Phosphate buffer saline is biological buffer, commonly used for research purpose. This buffer
maintains the proper or constant pH pf cell. It is prepared by pH-adjusted mixture of various
element. Here we prepare the PBS buffer from properly designed Tablet.

As working solution is 1X, therefore 1 tablet is designed to dissolve in 100 ml of distilled water.

1 tablet------------------------------------------------------100ml

5tablet -------------------------------------------------------500ml

Add 5 tablet in reagent bottle and mix it distilled water until proper dissolution. Now,
autoclave the solution. Store at 40C

Mouse Dissection
We slaughter the mouse for isolation of hematopoietic stem cell from femur, because the
concentration of Bone marrow is higher in this region.

Procedure

 Calm the mouse by rubbing it skin, then hold it tail and capture it from neck.
 Place it in chloroform, wait until it become unconscious.
 Kill the mouse via cervical dislocation because this is least painful method.
 Place the mouse in anterior posture, pick the skin from the middle and cut directly above
diaphragm.
 Now, it may be cut upward and downward, gradually isolate the organ and place them
into formaldehyde for preservation.
 Remove the femur bone, isolate the stem cell with help of syringe filled with PBS.
 You may place the cell in PBS for short period of time or directly transfer into media.
 Incubate it in CO2 incubator until confluency.