Mike Barnkob:Protocols/Immunology/Enrichment from tumours: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
No edit summary
Line 3: Line 3:
==T cell enrichment from tumours==
==T cell enrichment from tumours==


Protocol on how to isolate CD3+ T cells from ''in vivo'' tumours using bead separation.
Protocol on how to isolate T cells from ''in vivo'' tumours using percoll seperation.
 
'''NOT DONE'''


===Reagents and equipment===
===Reagents and equipment===


For harvesting tumour and lymph nodes:
For harvesting tumour and lymph nodes:
# Petri-dish
# Petri-dish.
# [[Mike_Barnkob:Protocols/Mediums|R0 media]] (kept in tissue-culture room).
# [[Mike_Barnkob:Protocols/Mediums|R10 media]].
# 70% ethanol.
# 70% ethanol.
# [[Mike_Barnkob:Protocols/Mediums|1x Collagenase Solution]] (kept in -20 freezer).


For single-cell suspension:
For single-cell suspension:
# GentleMACS Dissociator.
# C tubes for GentleMACS Dissociator.
# 50 ml Falcon tube  
# 50 ml Falcon tube  
# 70μM cell strainer  
# 70μM cell strainer  
# 2.5ml syringe
# 2.5ml syringe
# Red blood cell lysis buffer (Qiagen).
# [[Mike_Barnkob:Protocols/Mediums|R10 T cell media]].
# [[Mike_Barnkob:Protocols/Mediums|R10 T cell media]].


For T-cell enrichment / bead-seperation:
For Percoll seperation:
#  
# "100%" Percoll: Add 100 ml 10x PBS to 900 ml Percoll.
# Prepare dilutions of 80%, 50% and 40% solutions of "100%" Percoll, with 4 ml per dilution per sample.
 
For T-cell enrichment:
# Antibodies for FACS.


===Protocol===
===Protocol===


'''Harvest tumour:'''
'''Harvest tumour:'''
# Add 3 ml 1x Collagenase solution to each gentleMACS C tube, keep on ice.
# Euthanize mouse by cervical dislocation.
# Euthanize mouse by cervical dislocation.
# Dissect out tumour<br />'''Note:''' Wet large area of skin with 70% ethanol to disinfect.<br />'''Note:''' Keep hair out of incision.  
# Dissect out tumour and spleen<br />'''Note:''' Wet large area of skin with 70% ethanol to disinfect.<br />'''Note:''' Keep hair out of incision.  
# Using forceps, dissect out tumour.<br />'''Note:''' remove as much surrounding fat and skin as possible.
# Using forceps and scissor, dissect out tumour.<br />'''Note:''' remove as much surrounding fat and skin as possible, but keep skin on top.
# Place tumour in petri dish and weigh
# Place tumour in petri dish and weigh.
# Cut tumours into smaller pieces (2-4mm) and transfer into tube with R0 media and collagenase. Keep on ice.
# Cut tumours into smaller pieces (2-4mm) and transfer into tube with 5ml R10 media. Keep on ice.
# Dissect out spleen and transfer into tube with 5ml R10 media. Keep on ice.


'''Create single-cell suspension and lyse red blood cells:'''
'''Create single-cell suspension:'''
# Place gentleMACS tubes in dissociator and run using program '''m_impTumor_02'''.
# Add 1mL of R10 T cell media to 50 ml Falcon tube. Place 70μM cell strainer on tube and transfer tumour onto cell strainer.<br />'''Note:''' Work in tissue-culture hood.
# Place gentleMACS tubes in 37°C incubator for 30 minutes. Mix by inverting tubes every 10 minutes.
#  Take out plunger of 2.5ml syringe, and mesh tumour through strainer with blunt end of the plunger.<br />Note: Flush cells through with a little media afterwards.<br />Note: When done, add up to 10-15 ml R10 T cell media total.
# Place gentleMACS tubes in dissociator and run using program '''m_impTumor_03'''.
# Add 1mL of R10 T cell media to 50 ml Falcon tube. Place 70μM cell strainer on tube and transfer dissociated tumour onto cell strainer.<br />'''Note:''' Work in tissue-culture hood.
#  Take out plunger of 2.5ml syringe, and mesh tumour through strainer with blunt end of the plunger.<br />Note: Flush cells through with a little media afterwards.<br />Note: When done, add up to 5 ml R10 T cell media.
# Spin cells down at 1500 RPM for 5 min and remove supernatant.
# Spin cells down at 1500 RPM for 5 min and remove supernatant.
# Resuspend in 2 ml of red blood cell lysis buffer and leave on ice for 5 min.
# Optional step: resuspend in 2 ml of red blood cell lysis buffer and leave on ice for 3 min. Add 5 ml of media, spin cells down at 1500 RPM for 5 min and remove supernatant.
# Add 5 ml of media, spin cells down at 1500 RPM for 5 min and remove supernatant. 
# Resuspend in 1 ml R10 T cell media and count cells.
 
'''MACS bead seperation:'''
 
 
'''mRNA isolation:'''
 
''Follows Qiagen RNeasy Mini Kit instructions and presumes all reagents have been prepared.''
# (Spin cells down at 1500 RPM for 5 min and remove supernatant)
# Disrupt and homogenize:
#* Resuspend in 600μL buffer RLT and pipet up and down several times.
#* Transfer to 1.5 Eppendorf tube
#* Centrifuge the lysate for 3 min at full speed and transfer the supernatant to new 1.5 Eppendorf tube.<br />'''Note:''' The lysate is normally between 250-600 μL.<br />'''Note:''' When adding RLT, liquid can become viscus, so pipet slowly.
#* Add 1 volume of 70% ethanol to the lysate, and mix immediately by pipetting (don't vortex).
# Transfer 700 μL of sample to an RNeasy spin column placed in a 2 ml collection tube. Close lid gently, and centrifuge for 15 s at 10,000 rpm. Discard the flow-through.
# Add 700 μL Buffer RW1 to the RNeasy spin column. Close lid gently, and centrifuge for 15 s at 10,000 rpm. Discard the flow-through.
# Add 500 μL Buffer RPE to the RNeasy spin column. Close lid gently, and centrifuge for 15 s at 10,000 rpm. Discard the flow-through.
# Add 500 μl Buffer RPE to the RNeasy spin column. Close lid gently, and centrifuge for 2 minutes at 10,000 rpm. Discard the flow-through.
# Place the RNeasy spin column in a new 1.5 ml collection tube. Add 30–50 μL RNase-free water directly to the spin column membrane. Close the lid gently, and centrifuge for 1 min at 10,000 rpm to elute the RNA.
# Label and store at -20°C.


'''Quality control:'''<br />
'''Percoll seperation:'''
''Using a NanoDrop''
# Create Percoll gradient: Add 80% solution to bottom of 15ml Falcon tube. Slowly add 50% solution on top.<br/>'''Note:''' I normally use 4 ml per gradient.
# A260/A280: ratio should be close to 1.8-2.0 [http://en.wikipedia.org/wiki/Nucleic_acid_quantitation].
# Resuspend samples in the 40% solution, and very slowly add to 80%-50% tubes. Draw lines on the tubes of where the gradients are.
# A260/A230: ratio should be close to 2.0 when isolating low amounts of RNA. Values < 1.0 indicates contamination with salts or rests of phenol or protein in the RNA solution.
# Spin samples at 400g for 30 minutes with lowest speed-up and speed-down speeds.
''Gel:''
# Using 5ml pipet, transfer dead cells from top of samples and discard.
# Make [[Mike_Barnkob:Protocols/Cloning/Gels|1% agarose gel]]
# Using Pastur-pipet aspirate interfase at 80%-50% border and transfer to new 15ml Falcon tube.<br/>'''Note:''' Starting with the spleen sample, it should be easy to see where the lymphocyte population is. If nothing is visible, aspirate the whole 50% gradient.
# Mix in Eppendorf tube:
# Wash cells in either FACS buffer or R10 for T-cells media and count.
#* 4 μL dH20
#* 5 μL Blue Loading Dye (6X)
#* 1 μL RNA sample
# Run the gel at 80 V until the fastest dye has moved 2/3 of the gel length (90-100 minutes)
# Visualize gel using a UV transilluminator. A good extraction should have two clearly distinguishable bands, like below:
[[Image:Mike_Barnkob_2015-01-22_mRNA_gel.jpg]]


'''Storage:'''
'''Futher processing:'''
* Purified RNA may be stored at –20°C or –70°C in RNase-free water. Under these conditions, no degradation of RNA is detectable after 1 year.
# Stain cells for FACS or bead-seperation.


===References===
===References===

Revision as of 14:11, 30 April 2015

← Front page

T cell enrichment from tumours

Protocol on how to isolate T cells from in vivo tumours using percoll seperation.

Reagents and equipment

For harvesting tumour and lymph nodes:

  1. Petri-dish.
  2. R10 media.
  3. 70% ethanol.

For single-cell suspension:

  1. 50 ml Falcon tube
  2. 70μM cell strainer
  3. 2.5ml syringe
  4. R10 T cell media.

For Percoll seperation:

  1. "100%" Percoll: Add 100 ml 10x PBS to 900 ml Percoll.
  2. Prepare dilutions of 80%, 50% and 40% solutions of "100%" Percoll, with 4 ml per dilution per sample.

For T-cell enrichment:

  1. Antibodies for FACS.

Protocol

Harvest tumour:

  1. Euthanize mouse by cervical dislocation.
  2. Dissect out tumour and spleen
    Note: Wet large area of skin with 70% ethanol to disinfect.
    Note: Keep hair out of incision.
  3. Using forceps and scissor, dissect out tumour.
    Note: remove as much surrounding fat and skin as possible, but keep skin on top.
  4. Place tumour in petri dish and weigh.
  5. Cut tumours into smaller pieces (2-4mm) and transfer into tube with 5ml R10 media. Keep on ice.
  6. Dissect out spleen and transfer into tube with 5ml R10 media. Keep on ice.

Create single-cell suspension:

  1. Add 1mL of R10 T cell media to 50 ml Falcon tube. Place 70μM cell strainer on tube and transfer tumour onto cell strainer.
    Note: Work in tissue-culture hood.
  2. Take out plunger of 2.5ml syringe, and mesh tumour through strainer with blunt end of the plunger.
    Note: Flush cells through with a little media afterwards.
    Note: When done, add up to 10-15 ml R10 T cell media total.
  3. Spin cells down at 1500 RPM for 5 min and remove supernatant.
  4. Optional step: resuspend in 2 ml of red blood cell lysis buffer and leave on ice for 3 min. Add 5 ml of media, spin cells down at 1500 RPM for 5 min and remove supernatant.

Percoll seperation:

  1. Create Percoll gradient: Add 80% solution to bottom of 15ml Falcon tube. Slowly add 50% solution on top.
    Note: I normally use 4 ml per gradient.
  2. Resuspend samples in the 40% solution, and very slowly add to 80%-50% tubes. Draw lines on the tubes of where the gradients are.
  3. Spin samples at 400g for 30 minutes with lowest speed-up and speed-down speeds.
  4. Using 5ml pipet, transfer dead cells from top of samples and discard.
  5. Using Pastur-pipet aspirate interfase at 80%-50% border and transfer to new 15ml Falcon tube.
    Note: Starting with the spleen sample, it should be easy to see where the lymphocyte population is. If nothing is visible, aspirate the whole 50% gradient.
  6. Wash cells in either FACS buffer or R10 for T-cells media and count.

Futher processing:

  1. Stain cells for FACS or bead-seperation.

References

Retrieved from "https://openwetware.org/mediawiki/index.php?title=Mike_Barnkob:Protocols/Immunology/Enrichment_from_tumours&oldid=882975"