Arichard:Protocols/neuronalnucleiisolation20131203: Difference between revisions

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#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

  1. Place brain samples in the cryostat for ~20-30 minutes prior to beginning
  2. 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.
  3. 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.
  4. 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.
  5. Pre-wet filter with PBSE. Filter suspension, wash with 2 mls of PBSE.
  6. Spin down 5 mins at 1700 RPM
  7. 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.

  1. Spin down 5 mins 1700 RPM and aspirate supernatant.
  2. 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.
  3. Spin down 5 mins at 1700 RPM, Aspirate, Wash in PBSE, Spin, Aspirate.
  4. Resuspend in Blocking Buffer for 15-45 mins.
  5. Add Primary Antibody (NeuN) and RNase for 20 mins. (or over night).
  6. Without spinning, add ~3 volumes of PBSE, Spin, Aspirate, Wash, Spin, Aspirate.
  7. Add Secondary antibody + DNA dye. If there is time leave ~1 hr. If time sensitive – leave in for sorting.