Process of Culturing

Process of culturing
First phase
1. Take up your gloves take the PLL from refrigerator 0.5mg/μl
2. go to heat bar aseptic principle open and set button
3. dish 4X6 plates
4. using water to wash PLL alcohol 70% to disinfect, also watch out the clean of operate
floor
5. big pipette press up button to absorb, down button to release 5ml
6. micropipette with pink tip to recycle to the empty falcon tube
7. 10 min waiting
8. 50ml＋200ml miliQ
9. Wash up by diluent 200Ul X 24 plates
10. 37 1 day waiting
11. Abandon the PLL milliQ wash (quantity X) via Mpipe
12. 2 hours waiting
13. PBS to dish wash and another 2 hours waiting (quantity X) via ?
14. NCM-HS 400ul to plates ----------(the making method of NCH-HS)
Second phase dissect mice

1. need neuro medium 242 NCH HS red one; antiobody(PS) glutamineX1
2. 242 heat in 37 up and down shake （NCH production is needed）
3. DB used in dissection dry heated in device
4. DB in dish (3 dishes in 1 empty) 3ml/dish
5. Tools of dissection 2 short tweezers 1 long tweezer 1 disinfect scissor
6. Prepare crushed ice under water heater and plastic bag
7. Do sugery------ dissection
8. Aspirate neuro cells gently after Trpsin’s reaction in heat water buffer
RNA purification
Preparation of Lysis and Binding Solution Add 1/100 volume of 2-ME. Bring Wash Solutions
I and II to room temperature.
Remove culture medium from the well, add 300 uL of Lysis & Binding Solution
(containing 2-ME), and freeze at -80°C.
Dissolve in a 25°C heat block, return to room temperature, and mix thoroughly by pipetting
with an RNAseFree P1000 tip.
Transfer to a 1.5 mL tube. It is better to use a DNA low-binding tube.
Vortex for 30 sec, then incubate at room temperature for 10-15 min. It seems better to use
a bead crusher at 2430 rpm for 10 sec. In some cells, RNA yield may dramatically improve.
Add 35 uL of Magnetic Bead Solution. Vortex the magnetic beads to make a uniform
suspension before adding.
Vortex for 20 s.
Separate using a magnet.
Place 6 tubes in the magnet holder, press down the lid, and invert 2-3 times. This will
remove
magnetic beads attached near the lid. Alternatively, a weak centrifugation (7000 rpm, 5 sec)
is also acceptable.
Rotate the tube 2-3 times in the holder to accumulate the beads. Beads will form a circle
slightly above the bottom.
Using an aspirator with an RNase Free tip (P10), remove the liquid from the opposite side
of
the wall where the magnet is attracting the beads. Also, remove the supernatant remaining
on
the inner side of the lid and the wall near the top of the tube. You can use the same tip for
all
samples.
Add 250 uL of Wash Solution I and wash. Add 300 uL of Wash Solution II and wash 2
times.Separate using a magnet, discard the supernatant.Spin at 15000 rpm for 10 sec with
an empty tube.
Press the pipette tip firmly against the bottom to completely remove the liquid. Try
not to aspirate the beads.Add 35 uL of Elution Solution.Vortex for 10 s.Briefly spin down.
Or, flick off the beads by hand. Incubate at 65°C for 2 min.Vortex for 10 s. Spin at 15000
rpm for 10 sec.
RT-PCR and Q-PCR
RT-PCR
Prepare the mix in tubes with 4MM,water and RNA mix by pipetting.
Reaction in PCR Machine (Removal of genome DNA): 37℃ 5 min → 12℃ 1 min
Prepare the mix in tubes of 5II MM GS(LV) or GAS(AAV) primers and mix by pipetting.
Reaction in PCR Machine (Reverse transcription - Inactivation of reverse transcription
enzyme):
37℃ 15 min → 42℃ 5 min → 50℃ 5 min → 98℃ 5 min → 12℃
Dilution with MilliQ H2O
Q-PCR
Prepare ref and rep standard solution and wait samples melted
Mix well Gtaqmix with water and primers of REF and REP gene. Via Machine pipette
divides mixture into bottom of QPCR plate, take care surplus so we need calculate more
times than standard plus sample groups.
Create standard range groups, Add MilliQ water in tubes: 540, 270, 270, 270, 270, 270, 270
uL. Add 2.7 uL of 1 mg/mL midiprep solution to tube 1 and vortex well. Transfer 2.7 uL from
tube 1 to 2 and vortex well. Transfer 30 uL from tube 2 to 3 and vortex well. Sequentially
transfer 30 uL to tube 7 and vortex well.
Apply 2 uL from tubes 3 to 7 to the wells. Apply in order from the most diluted solution,
Once all applications are completed, attach a transparent film to the entire plate. Remove
the seal and place it on the plate. Press down from the top using a dedicated spatula.
Press the corners of the wells firmly in all directions to ensure proper adhesion. Trim any
excess film from the left and right perforations.
Set the plate in a centrifuge and spin for approximately 1 minute. After centrifugation, tap
the plate against the table several times to mix. Spin again.
Immunofluorescence
1.Fixation: remove medium, use PFA/PBS 4% 400ul each well PFA need to be water
buffered
20 mins in room temperature
2.remove PFA, add PBST 400ul
3.0.25 TritionX PBS 400ul, 20 mins in room temperature
4.remove solution, blocking solution BSA 200 ul, 20 mins in room temperature
5.Add primary antibody, watch out dilution times
6.need to shake, incubate in either room temperature for 2 hours or 4°C for 16 hours.
7.Wash: Remove the solution and add 400-600 µl of PBS-Tween. Wait for 5 minutes.
Repeat the above wash step three times.
8.Adjust the secondary antibody + DAPI to 220 µl/well in PBS (measure as a final 220 µl).
Remove the solution and add 20 µl of the diluted secondary antibody to each well. Shake
the plate side to side to mix well. Incubate at room temperature for 1 hour.
9. Wash: Remove the solution and add 400-600 µl of PBS-Tween. Wait for 5 minutes.
Repeat the above wash step three times.
10. Remove the solution and wash once with MilliQ water. Then, drop 10 µl of mounting
medium. Gently place a coverslip (15mm diameter) using tweezers. If any bubbles form,
gently press down with tweezers to release the bubbles.

Process of Culturing

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Process of Culturing

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Process of culturing

Second phase dissect mice

2.remove PFA, add PBST 400ul

3.0.25 TritionX PBS 400ul, 20 mins in room temperature

5.Add primary antibody, watch out dilution times

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