Protocols

Protocol:  Mycoplasma Detection Kit and Culturing HEK-2 cells 
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Created by PI on 2014-03-20
# Notes:
    1. Protocol has been taken from PlasmoTest Mycoplasma Detection Kit user manual 
    2. Be sure to work in a sterile environment at all times 
# Materials:
    1. 15-mL polystyrene conical tube
    2. 50-mL polystyrene conical tubes
    3. Cryogenic Vials, Corning Incorporated, 2-mL, Lot# 34308068, Cat# 430488, 
    4.	T-25 cm2 Flask
        1.Thermo Scientific, BioLite 75 cm2 Flask Vented, Lot#A5RA5NG113, Cat#130190
    5.	T-175 cm2 Flask
        1.Thermo Scientific, BioLite 175 cm2 Flask Vented, Lot#C4WA4PE112, Cat#130191
    6.	HEK-2 Cell Line, Location: 1st floor freezer
        1.Be sure the cells are P30 or lower, otherwise they are NOT viable for testing
    7.	Growth Medium, prepared on 2012-07-30 by SP
        1.DMEM: HyClone, Cat# SH30285.01, Lot# AXF39350
        2.	Fetal Bovine Serum (FBS), Location: refrigerator #5
        3.	Pen/Strep: Prepared by N/A on N/A, Location: refrigerator #5
        4.	L-Glutamine: Prepared by N/A on N/A, Location: refrigerator #5
        5.	1 X Normacin: Invivogen, Cat# ant-nr-1, ant-nr-2 
        6.	1 X HEK-Blue Selection: Invivogen, Cat# hb-sel 
    8.	Sterile Phosphate Buffered Saline (PBS) 1x, prepared on N/A by N/A
    9.	Freeze down solution, 90% Growth medium, 10% (v/v) Dimethyl Sulfoxide (DMSO)
        1.	Fetal Bovine Serum (FBS): Prepared by N/A on N/A
        2.	Dimethyl Sulfoxide, Research Organics, Cat# 2166D, Lot# A85277, Received: Jan. 2010, Location: cell culturing room
    10.	Jouan CR 412 Centrifuge
    11.	Incubator


# Methods: 
Thawing Cells
    1.	Pipet sufficient volume of growth medium into 50-mL conical vial(s) and place medium into 37 degrees C/5% CO2 incubator for at least 30 minutes before use. 
    2.	Pipet 15-mL of warm growth medium into a 50-mL conical vial. Thaw cells in 37 degree water bath until cell/ice pellet loosens from tube wall and quickly transfer cell/ice pellet to 1-mL of warm growth medium. Add 9-mL of warm growth medium to vial for total volume of approximately 11-mL. Gently invert tube several times.
    3.	Remove 10-uL of cells in growth medium and pipet into 0.5-mL microcentrifuge tube for cell count
    4.	Add 10-uL of Trypan Blue to the 10-uL aliquot of cells and mix well with pipet. Add 10-uL of mixture into cell counter.
        a.	Record cell counts and show calculations:
    5.	After counting cells, centrifuge original cell volume at 1500 RPM at 4 degrees C for 5 minutes.
    6.	Remove medium from cells being careful not to disturb the cell pellet and add appropriate volume of fresh warm growth medium. 
    7.	Add the cells to a T-25 cm2  flask. Leave in the incubator overnight.  
    8.	Split the cells the next day. Cells should be seeded between 5x106 and 1x107 cells/cm2 in a T-175 flask. Flask size used will be determined on cell count. 
    9.	Change medium 2 times a week. 
Splitting Cells
    1.	Pipet sufficient volume of growth medium into 50-mL conical vial(s) and place medium into 37 degrees C/5% CO2 incubator for at least 30 minutes before use. A vial of sterile PBS should be placed in the incubator with the medium after thawing.
    2.	Remove growth medium from cell culture flasks with a sterile pipet and suction apparatus. Add warm PBS to each flask and remove the cells by tapping and using a cell scraper. 
    3.	Pipet PBS cells into a conical vial and centrifuge at 1500 RPM at 4 degrees C for 5 minutes
    4.	Remove medium from cells being careful not to disturb the cell pellet and add appropriate volume of fresh warm growth medium. Cells should be seeded between 5x106 and 1x107 cells/cm2 in a T-175 flask. Flask size used will be determined on cell count.
    5. BE SURE TO RECORD PASSAGE NUMBER. Note: cells are not viable after 30 passages and it is important to keep track.  
    6.	Change medium 2 times a week. 

Freezing Cells
    1.	Spin resuspended cells at 1500 RPM for 5 minutes at 4 degrees C.
    2.	Remove medium from cells being careful not to disturb the cell pellet and add appropriate volume of cold freeze down solution to obtain 5 or 10 million cells/mL in the cryogenic vial. Be sure to properly mix resuspended cells to ensure proper freeze down concentration.
    3.	Aliquot 1-mL of cells in freeze down solution in cryogenic vial(s). Work quickly as DMSO is toxic to cells.
    4.	Place cryogenic vial(s) in -80 degrees C freezer tray overnight.
    5.	Move cryogenic vial(s) to -150 degrees C freezer for storage the following day. Be sure to update the record folder.
Mycoplasma Detection: 
    1.	Collect 500 μL of supernatant of cell culture to be tested and transfer into a 1.5mL tube. Scrape the bottom of the dish to collect some cells. 
    2.	Boil or heat all the samples in a water bath of heating block for 15 min at 100°C. Let the samples cool down for a few minutes at room temperature. 
    3.	Add 50 μL of each sample, 1 X positive control, and 1 X negative control per well of a flat-bottom 96-well plate. 
    4.	Rinse HEK-2 cells with warm PBS. 
    5.	After rinse, add warm PBS to detach cells using a cell scraper. Estimate the cell count as previously noted. 
    6.	Dilute the cells with warm HEK-Blue Detection medium at a concentration of 1 - 3.5 x 105 cells/mL. DO NOT add more because it may result in a loss of sensitivity of the kit. 
    7.	 Add 200 μL of cell suspension into each well containing a sample, controls, and one without any sample
    8.	incubate the plate at 37 degrees C/5% CO2   for 16 - 24 hours 
    9.	Positive control should appear purple or blue, the negative control should be pink or light purple, and the well containing only cell suspension should appear pink or light purple.