Alac2: /* Cell Culture */

2021-02-04T19:55:37Z

Cell Culture

← Older revision		Revision as of 19:55, 4 February 2021
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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:		#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~~<br>~~		#*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		#*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. ''  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. ''  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. ''  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.

	<br>		<br>
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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. '' '' ''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.

	<br>		<br>

Admin: Created page with "= Protocol = ===== Cell Culture
===== #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..."

2021-02-01T19:36:57Z

Created page with "= Protocol = ===== Cell Culture ===== #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..."

New page

= Protocol =

===== Cell Culture =====

#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. 
#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. 
#Add the cells to the six well plate at 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
#Rock the plate sideways to evenly distribute the cells.
#Culture the cells overnight at 37oC. 

 

===== Transfection =====

#Thaw the DNA to be used for transfection. 
#Set up a reaction for each well in a microcentifuge tube:
#*Add 4.0 µg of DNA to 200 µl of serum-free media. Vortex the tube for 1-2 seconds to mix.
#*Add 10 µl of the Fugene-HD Transfection Reagent. Flick the tube 15-20 times to mix. ''  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. 
#Let the reaction sit for 20 min at Rm temp in the tissue culture hood, for the DNA-containing micelles to form. 
#Add the total reaction volume of each microcentrifuge tube to its respective cell well, drop-by-drop. Shake the plate sideways to mix. 
#Incubate the transfecting cells overnight at 37oC.

 

===== Paraformaldehyde Cell Fixation =====

#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.
#Set up a batch reaction. For six wells, combine:
#*1.2 ml 10X PBS 
#*3 ml 18% PFA 
#*10.8 ml ddH2O
#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.

J774 Cell Transfection - Revision history

Alac2: /* Cell Culture */

Admin: Created page with "= Protocol = ===== Cell Culture ===== #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..."

Admin: Created page with "= Protocol = ===== Cell Culture
===== #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..."