Arichard:Protocols/neuronalnucleiisolation20131203: Difference between revisions
Jump to navigation
Jump to search
>Andrew (Created page with "Created 9/17/13 by GEK Mixed Diane and Yun’s Protocols Human Nuclei Isolation and NeuN Staining for FCM/FACS #Place brain samples in the cryostat for ~20-30 minutes prior ...") |
>Andrew No edit summary |
||
Line 5: | Line 5: | ||
#Place brain samples in the cryostat for ~20-30 minutes prior to beginning | #Place brain samples in the cryostat for ~20-30 minutes prior to beginning | ||
#While working in the cryostat, isolate 0.15-0.5g of tissue using a razor blade in a weigh boat. Cut into smaller pieces in the weigh boat. | #While working in the cryostat, isolate 0.15-0.5g of tissue using a razor blade in a weigh boat. Cut into smaller pieces in the weigh boat. | ||
#Place tissue into 2 ml of cold CMF (HBSS or PBS +2 mM EGTA) (Calcium Magnesium Free) – Cut into smaller pieces with either forceps or the razor blade. Once minced, let sit for ~30 minutes. | #Place tissue into 2 ml of cold CMF (HBSS or PBS +2 mM EGTA) (Calcium Magnesium Free) – Cut into smaller pieces with either forceps or the razor blade. Once minced, let sit for ~30 minutes. | ||
#Triturate the cells with 3 different bore size tips. (Cut them with a razor blade - ~4mm and 2 mm, and normal p1000 tip). Cells should be close to a single cell solution. | #Triturate the cells with 3 different bore size tips. (Cut them with a razor blade - ~4mm and 2 mm, and normal p1000 tip). Cells should be close to a single cell solution. | ||
#Pre-wet filter with PBSE. Filter suspension, wash with 2 mls of PBSE. | #Pre-wet filter with PBSE. Filter suspension, wash with 2 mls of PBSE. | ||
#Spin down 5 mins at 1700 RPM | #Spin down 5 mins at 1700 RPM | ||
#Aspirate supernatant, add 5 mls of PBSE+1% NP40 to lyse the cells. Add slowly while vortexing gently. Sit for 10 mins on ice with vortexing | #Aspirate supernatant, add 5 mls of PBSE+1% NP40 to lyse the cells. Add slowly while vortexing gently. Sit for 10 mins on ice with vortexing | ||
every 2 mins. | every 2 mins. | ||
#Spin down 5 mins 1700 RPM and aspirate supernatant. | #Spin down 5 mins 1700 RPM and aspirate supernatant. | ||
#Add 100ul PBSE to resuspend pellet. Add 4 mls of 70% (cold) ETOH and sit 30 mins (for sequencing work). Add 4 mls of 2-4% PFA for other applications. Skip this step for transcriptome work. | #Add 100ul PBSE to resuspend pellet. Add 4 mls of 70% (cold) ETOH and sit 30 mins (for sequencing work). Add 4 mls of 2-4% PFA for other applications. Skip this step for transcriptome work. | ||
#Spin down 5 mins at 1700 RPM, Aspirate, Wash in PBSE, Spin, Aspirate. | #Spin down 5 mins at 1700 RPM, Aspirate, Wash in PBSE, Spin, Aspirate. | ||
#Resuspend in Blocking Buffer for 15-45 mins. | #Resuspend in Blocking Buffer for 15-45 mins. | ||
#Add Primary Antibody (NeuN) and RNase for 20 mins. (or over night). | #Add Primary Antibody (NeuN) and RNase for 20 mins. (or over night). | ||
#Without spinning, add ~3 volumes of PBSE, Spin, Aspirate, Wash, Spin, Aspirate. | #Without spinning, add ~3 volumes of PBSE, Spin, Aspirate, Wash, Spin, Aspirate. | ||
#Add Secondary antibody + DNA dye. If there is time leave ~1 hr. If time sensitive – leave in for sorting. | #Add Secondary antibody + DNA dye. If there is time leave ~1 hr. If time sensitive – leave in for sorting. |
Latest revision as of 21:22, 4 December 2013
Created 9/17/13 by GEK Mixed Diane and Yun’s Protocols
Human Nuclei Isolation and NeuN Staining for FCM/FACS
- Place brain samples in the cryostat for ~20-30 minutes prior to beginning
- While working in the cryostat, isolate 0.15-0.5g of tissue using a razor blade in a weigh boat. Cut into smaller pieces in the weigh boat.
- Place tissue into 2 ml of cold CMF (HBSS or PBS +2 mM EGTA) (Calcium Magnesium Free) – Cut into smaller pieces with either forceps or the razor blade. Once minced, let sit for ~30 minutes.
- Triturate the cells with 3 different bore size tips. (Cut them with a razor blade - ~4mm and 2 mm, and normal p1000 tip). Cells should be close to a single cell solution.
- Pre-wet filter with PBSE. Filter suspension, wash with 2 mls of PBSE.
- Spin down 5 mins at 1700 RPM
- Aspirate supernatant, add 5 mls of PBSE+1% NP40 to lyse the cells. Add slowly while vortexing gently. Sit for 10 mins on ice with vortexing
every 2 mins.
- Spin down 5 mins 1700 RPM and aspirate supernatant.
- Add 100ul PBSE to resuspend pellet. Add 4 mls of 70% (cold) ETOH and sit 30 mins (for sequencing work). Add 4 mls of 2-4% PFA for other applications. Skip this step for transcriptome work.
- Spin down 5 mins at 1700 RPM, Aspirate, Wash in PBSE, Spin, Aspirate.
- Resuspend in Blocking Buffer for 15-45 mins.
- Add Primary Antibody (NeuN) and RNase for 20 mins. (or over night).
- Without spinning, add ~3 volumes of PBSE, Spin, Aspirate, Wash, Spin, Aspirate.
- Add Secondary antibody + DNA dye. If there is time leave ~1 hr. If time sensitive – leave in for sorting.