Splitting Cells: Difference between revisions

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==SOP==

*[[SOP-_Biosafety Cabinet|SOP- Biosafety Cabinet]]

*[[SOP-_Cryogenic Materials|SOP- Cryogenic Materials]]

*[[SOP-_Vacuum Pumps|SOP- Vacuum Pumps]]

==Materials==

*Media (~~L1-FBS for 3T3-L1, COS-~~FBS ~~for others~~): Prepare by filtering together 5 mL PSG, 50 mL COS-FBS and one bottle of DMEM

*Media (FBS or NCS as required): Prepare by filtering together 5 mL PSG, 50 mL COS-FBS and one bottle of DMEM

(To prepare DMEM:

1 bottle of DMEM found in 4c in cell culture room

1 tube FBS found in -80 in cell culture room

5 mLs PSG found in door of 4c in cell culture room

500 mL bottle taken from lab

filter found on metal rack behind fume hood

•pour all ingredients into DMEM bottle

•screw filter onto 500 mL bottle

•attach hose onto arm of filter

•pour mixture into filter)

*PBS -/-

*0.05% Trypsin

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==Protocol==

#Warm PBS and Media in water bath

#Wash cells ~~twice~~ with 10 mL (per 10 cm dish) PBS -/-

# Aspirate the plate media

#Add 1 mL trypsin and sit in the hood for 2-5 min

#Wash cells once with 10 mL (per 10 cm dish) PBS -/- then aspirate the PBS

#Add 1 mL trypsin and allow to sit in the hood for 2-5 min

#Add 10 mL media to each new dish

#Check cells for trypsinization, and if necessary tap the cells

#Add 9 mL media to trypsinized cells

#Add 1 mL cells to each dish (for 10X dilution; 0.5 mL for 20X)

#Replace plates in the incubator

#Replace plates in the 37C incubator

==Cell Specific Notes==

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+==SOP==
+*[[SOP-_Biosafety Cabinet|SOP- Biosafety Cabinet]]
+*[[SOP-_Cryogenic Materials|SOP- Cryogenic Materials]]
+*[[SOP-_Vacuum Pumps|SOP- Vacuum Pumps]]
 ==Materials==
-*Media (L1-FBS for 3T3-L1, COS-FBS for others):  Prepare by filtering together 5 mL PSG, 50 mL COS-FBS and one bottle of DMEM
+*Media (FBS or NCS as required):  Prepare by filtering together 5 mL PSG, 50 mL COS-FBS and one bottle of DMEM
+(To prepare DMEM:
+bottle of DMEM found in 4c in cell culture room
+tube FBS found in -80 in cell culture room
+mLs PSG found in door of 4c in cell culture room
+mL bottle taken from lab
+filter found on metal rack behind fume hood
+•pour all ingredients into DMEM bottle
+•screw filter onto 500 mL bottle
+•attach hose onto arm of filter
+•pour mixture into filter)
 *PBS -/-
 *0.05% Trypsin
@@ Line 6: / Line 21: @@
 ==Protocol==
 #Warm PBS and Media in water bath
-#Wash cells twice with 10 mL (per 10 cm dish) PBS -/-
+# Aspirate the plate media
-#Add 1 mL trypsin and sit in the hood for 2-5 min
+#Wash cells once with 10 mL (per 10 cm dish) PBS -/- then aspirate the PBS
+#Add 1 mL trypsin and allow to sit in the hood for 2-5 min
 #Add 10 mL media to each new dish
 #Check cells for trypsinization, and if necessary tap the cells
 #Add 9 mL media to trypsinized cells
 #Add 1 mL cells to each dish (for 10X dilution; 0.5 mL for 20X)
-#Replace plates in the incubator
+#Replace plates in the 37C incubator
 ==Cell Specific Notes==