J774 Cell Transfection: Difference between revisions

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(Created page with "= Protocol = ===== Cell Culture<br> ===== #Prepare a 6-well plate by adding 2 ml of J774 Cell media per well. If coverslips are required, add one coverslip/well prior to add...")
 
 
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===== Cell Culture<br> =====
===== Cell Culture<br> =====


#Prepare a 6-well plate by adding 2 ml of J774 Cell media per well. If coverslips are required, add one coverslip/well prior to adding the media.<br>  
#Prepare a 12-well plate by adding 1 mL of fresh DMEM per well. If coverslips are required, add one coverslip per well prior to adding media.<br>
#Wash the cells pre-cultured in a T25 flask with 5 ml of PBS and trypsinize with 1.5 ml of trypsin for five minutes. After adding 3.5 ml of fresh media, determine the cell concentration using a hemocytometer.<br>  
#Wash the cells pre-cultured in a T25 flask with 5 ml of PBS, then remove the PBS. Add 1 mL of trypsin for 5 min at 37<sup>o</sup>C. Add 4 mL of fresh media to deactivate the trypsin, then determine the cell concentration using a hemocytometer.<br>
#Add the cells to the six well plate at the following concentration:&nbsp;&nbsp;  
#Add the cells to the 12-well plate according to the following concentration:&nbsp;&nbsp;  
#*500,000 cells/well for subsequent microscopy analysis of transfected cells grown on the coverslips =&gt; ~35-40 cells/field-of-view at 63x, with ~20% transfected<br>
#*500,000 cells/well for subsequent microscopy analysis of transfected cells grown on the coverslips =&gt; ~35-40 cells/field-of-view at 63x, with ~20% transfected
#*1 million cells/well for subsequent parachuting of cells onto coverslips   
#*1 million cells/well for subsequent parachuting of cells onto coverslips   
#Rock the plate sideways to evenly distribute the cells.  
#*Alternatively, if cells are 80-90% confluent, add 3-5 drops of trypsinzed cells into each well
#Rock the plate sideways to evenly distribute the cells.
#Culture the cells overnight at 37<sup>o</sup>C.<br>  
#Culture the cells overnight at 37<sup>o</sup>C.<br>  


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#Thaw the DNA to be used for transfection.<br>  
#Thaw the DNA to be used for transfection.<br>  
#Set up a reaction for each well in a microcentifuge tube:  
#Set up a reaction for each well in a microcentrifuge tube:  
#*Add 4.0 µg of DNA to 200 µl of serum-free media. Vortex the tube for 1-2 seconds to mix.  
#*Add 3.3 µg of DNA to 150 µL of serum-free media. Vortex the tube briefly to mix.
#*Add 10 µl of the Fugene-HD Transfection Reagent. Flick the tube 15-20 times to mix. ''&nbsp; Note:'' The Tranfection Reagent is ethanol-based and evaporates easily. Keep the Transfection Reagent in the fridge until its addition and do not leave uncapped. Re-wrap in parafilm after use.<br>  
#*Add 10 µL of FuGENE-HD Transfection Reagent. Flick the tube 15-20 times to mix. ''&nbsp; Note:'' The transfection reagent is ethanol-based and evaporates easily. Keep it in the fridge until its addition and do not leave uncapped. Re-wrap in parafilm after use.<br>
#Let the reaction sit for 20 min at Rm temp in the tissue culture hood, for the DNA-containing micelles to form.<br>  
#Let the reaction sit for 15 min at room temperature in the tissue culture hood, allowing the DNA-containing micelles to form. ''&nbsp; Note'': Do not exceed 20 min of incubation time. <br>
#Add the total reaction volume of each microcentrifuge tube to its respective cell well, drop-by-drop. Shake the plate sideways to mix.<br>  
#Add dropwise the total reaction volume of each microcentrifuge tube to its respective cell well. Rock the plate sideways to mix.<br>
#Incubate the transfecting cells overnight at 37<sup>o</sup>C.  
#Incubate the transfecting cells 24-48 hr at 37<sup>o</sup>C.


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===== Paraformaldehyde Cell Fixation<br> =====
===== Paraformaldehyde Cell Fixation<br> =====


#Aspirate the media. Wash the cells twice with non-sterile PBS, at 2mL/well. ''Note:'' Be careful not to knock the cells off the coverslips during the wash steps.  
#Aspirate the media. Wash the cells twice with non-sterile PBS, at 1 mL/well. ''&nbsp;'' ''Note:'' Be careful not to knock the cells off the coverslips during the wash steps.
#Set up a batch reaction. For six wells, combine:
#Add 1 mL of 4% PFA (diluted in PBS) into each well. Incubate on the plate shaker for 15 min at room temperature in the dark.
#*1.2 ml 10X PBS<br>
#Remove PFA and wash cells with 1 mL/well of non-sterile PBS for 5 min.
#*3 ml 18% PFA<br>
#Cover cells with 1 ml PBS/well and keep plate at 4<sup>o</sup>C.
#*10.8 ml ddH<sub>2</sub>O 
#Add 2mL of reaction/well. Let the plate sit at Rm temp for 15 min. ''Note:'' Keep in the dark if transfected with a fluorophore.  
#Aspirate. Wash once-twice with PBS and cover with 2 ml PBS/well.  


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Latest revision as of 19:55, 4 February 2021

Protocol

Cell Culture
  1. Prepare a 12-well plate by adding 1 mL of fresh DMEM per well. If coverslips are required, add one coverslip per well prior to adding media.
  2. Wash the cells pre-cultured in a T25 flask with 5 ml of PBS, then remove the PBS. Add 1 mL of trypsin for 5 min at 37oC. Add 4 mL of fresh media to deactivate the trypsin, then determine the cell concentration using a hemocytometer.
  3. Add the cells to the 12-well plate according to the following concentration:  
    • 500,000 cells/well for subsequent microscopy analysis of transfected cells grown on the coverslips => ~35-40 cells/field-of-view at 63x, with ~20% transfected
    • 1 million cells/well for subsequent parachuting of cells onto coverslips
    • Alternatively, if cells are 80-90% confluent, add 3-5 drops of trypsinzed cells into each well
  4. Rock the plate sideways to evenly distribute the cells.
  5. Culture the cells overnight at 37oC.


Transfection
  1. Thaw the DNA to be used for transfection.
  2. Set up a reaction for each well in a microcentrifuge tube:
    • Add 3.3 µg of DNA to 150 µL of serum-free media. Vortex the tube briefly to mix.
    • Add 10 µL of FuGENE-HD Transfection Reagent. Flick the tube 15-20 times to mix.   Note: The transfection reagent is ethanol-based and evaporates easily. Keep it in the fridge until its addition and do not leave uncapped. Re-wrap in parafilm after use.
  3. Let the reaction sit for 15 min at room temperature in the tissue culture hood, allowing the DNA-containing micelles to form.   Note: Do not exceed 20 min of incubation time.
  4. Add dropwise the total reaction volume of each microcentrifuge tube to its respective cell well. Rock the plate sideways to mix.
  5. Incubate the transfecting cells 24-48 hr at 37oC.


Paraformaldehyde Cell Fixation
  1. Aspirate the media. Wash the cells twice with non-sterile PBS, at 1 mL/well.   Note: Be careful not to knock the cells off the coverslips during the wash steps.
  2. Add 1 mL of 4% PFA (diluted in PBS) into each well. Incubate on the plate shaker for 15 min at room temperature in the dark.
  3. Remove PFA and wash cells with 1 mL/well of non-sterile PBS for 5 min.
  4. Cover cells with 1 ml PBS/well and keep plate at 4oC.


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