Shreffler:Confocal Protocols: Difference between revisions
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== Overview == | |||
Collection of protocols used by [[User:Alexander D. Praslick|Alex]] for the [[Shreffler:Notebook/Basophil_Imaging|basophil imaging project]]. | |||
== Preparation of Polylysine Coated Slides == | |||
=== Materials === | |||
*12 well tissue culture plate | *12 well tissue culture plate | ||
*round cover glasses (i.e. "slides") | *round cover glasses (i.e. "slides") | ||
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*1X Poly-L-Lysine (1:10, Poly-L-Lysine:dH<sub>2</sub>O) | *1X Poly-L-Lysine (1:10, Poly-L-Lysine:dH<sub>2</sub>O) | ||
=== Procedure === | |||
# Wash circular cover glasses in a small petri dish filled with 70% ethanol. Transfer cover glasses individually to the wells of culture plate with clean forceps and allow to ''air dry.'' | # Wash circular cover glasses in a small petri dish filled with 70% ethanol. Transfer cover glasses individually to the wells of culture plate with clean forceps and allow to ''air dry.'' | ||
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# Let slides stand for 20 minutes before aspirating off excess Poly-L-Lysine solution. Let slides air dry under hood. Slides can be stored at room temperature until ready to use. | # Let slides stand for 20 minutes before aspirating off excess Poly-L-Lysine solution. Let slides air dry under hood. Slides can be stored at room temperature until ready to use. | ||
== Stimulation of Basophils with Anti - IgE and fMLP == | |||
=== Materials === | |||
=== Procedure === | |||
'''fMLP''' | |||
*For first dilution (1:100), add 1 μL fMLP to 99 μL RPMI. | *For first dilution (1:100), add 1 μL fMLP to 99 μL RPMI. | ||
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*Add 250 μL of the second solution to 250 μL of basophil cell solution and incubate for 30 minutes at 37 °C. | *Add 250 μL of the second solution to 250 μL of basophil cell solution and incubate for 30 minutes at 37 °C. | ||
=== RPMI: Unstimulated Control === | |||
*Add 250 μL RPMI to 250 μL basophil cell solution. | *Add 250 μL RPMI to 250 μL basophil cell solution. | ||
*Incubate for 30 minutes at 37 °C. | *Incubate for 30 minutes at 37 °C. | ||
'''Anti-IgE''' | |||
*Add 0.6 μL Anti-IgE to 300 μL RPMI | *Add 0.6 μL Anti-IgE to 300 μL RPMI | ||
*Add 250 μL of this solution to 250 μL of basophil cell solution and incubate for 30 minutes at 37 °C. | *Add 250 μL of this solution to 250 μL of basophil cell solution and incubate for 30 minutes at 37 °C. | ||
== Plate & Fix Basophils == | |||
=== Materials === | |||
*30 ml PBS (Phosphate Buffered Saline) | *30 ml PBS (Phosphate Buffered Saline) | ||
*30 ml 4% Paraformaldehyde in PBS (PFA-PBS); made from 5 ml stock PFA in 25 ml PBS | *30 ml 4% Paraformaldehyde in PBS (PFA-PBS); made from 5 ml stock PFA in 25 ml PBS | ||
=== Procedure === | |||
# Once basophil cells are extracted from PBMC and stimulated, spin cells down at 300g for 7 minutes. | # Once basophil cells are extracted from PBMC and stimulated, spin cells down at 300g for 7 minutes. | ||
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# Keep Cells on ice and in the dark until subsequent procedures are carried out. | # Keep Cells on ice and in the dark until subsequent procedures are carried out. | ||
== Preparing Slides for Ab Tagging == | |||
=== Materials === | |||
*15 mL PBS | *15 mL PBS | ||
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*15 mL Stain Buffer (1% bovine albumin in 2 mM EDTA in PBS) | *15 mL Stain Buffer (1% bovine albumin in 2 mM EDTA in PBS) | ||
=== Procedure === | |||
# Wash slides once with 1 mL PBS per well. | # Wash slides once with 1 mL PBS per well. | ||
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== mAb Staining == | |||
=== Materials === | |||
=== Procedure === | |||
# ''Overnight Staining'' with goat anti-CD203c (1:100 from 200 μg/mL stock); rabbit anti-CD107a (1:400 from 200 μg/mL) in staining buffer | # ''Overnight Staining'' with goat anti-CD203c (1:100 from 200 μg/mL stock); rabbit anti-CD107a (1:400 from 200 μg/mL) in staining buffer | ||
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# Stain with biotinylated donkey anti-goat (1:1000), incubate 1 hour. | # Stain with biotinylated donkey anti-goat (1:1000), incubate 1 hour. | ||
# Wash 3 times for 5 minutes with TxPBS | # Wash 3 times for 5 minutes with TxPBS | ||
#; ''Prepare CD63 Ab'': For every 0.5 | #; ''Prepare CD63 Ab'': For every 0.5 mL (1 SLIDE) add 4 μL mouse anti-CD63 (1:125) in 15 uL staining buffer, add 8 μL Zenon Alexa 594. Incubate 10 min at RT, add 8 μL Zenon blocking reagent. Incubate 10 min at RT. | ||
# Stain with anti-CD63 cocktail, with 1:125 dilution of strepavidin Alexa 488 (for CD203c), and 1:400 donkey anti-rabbit 647 (for CD107a). Incubate for 1 hour. | # Stain with anti-CD63 cocktail, with 1:125 dilution of strepavidin Alexa 488 (for CD203c), and 1:400 donkey anti-rabbit 647 (for CD107a). Incubate for 1 hour. | ||
# Wash 3 times for 5 minutes with TxPBS | # Wash 3 times for 5 minutes with TxPBS | ||
# Fix with 4% PFA. Incubate for 15 minutes. | # Fix with 4% PFA. Incubate for 15 minutes. | ||
# Aspirate excess PFA, and mount slides with DAPI Vectashield mounting medium. Store at 4 °C in the dark until acquisition. | # Aspirate excess PFA, and mount slides with DAPI Vectashield mounting medium. Store at 4 °C in the dark until acquisition. | ||
=== Plate Setup === | |||
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==Discussion== | |||
[[Talk:{{PAGENAME}}|discuss this protocol]] | |||
==References== | |||
<biblio> | |||
</biblio> | |||
==Contact== | |||
*Who has experience with this protocol? | |||
*[[Special:Emailuser/Alexandar D. Praslick|Email Alex Praslick through OpenWetWare]] | |||
[[Category:ShreffLab]] | |||
[[Category:Protocol]] |
Latest revision as of 07:59, 27 June 2009
Overview
Collection of protocols used by Alex for the basophil imaging project.
Preparation of Polylysine Coated Slides
Materials
- 12 well tissue culture plate
- round cover glasses (i.e. "slides")
- 70% Ethanol (aq)
- 1X Poly-L-Lysine (1:10, Poly-L-Lysine:dH2O)
Procedure
- Wash circular cover glasses in a small petri dish filled with 70% ethanol. Transfer cover glasses individually to the wells of culture plate with clean forceps and allow to air dry.
- Under the hood apply 100 μL of 1X Poly-L-Lysine solution to center of each slide.
- Let slides stand for 20 minutes before aspirating off excess Poly-L-Lysine solution. Let slides air dry under hood. Slides can be stored at room temperature until ready to use.
Stimulation of Basophils with Anti - IgE and fMLP
Materials
Procedure
fMLP
- For first dilution (1:100), add 1 μL fMLP to 99 μL RPMI.
- Second dilution, take 3 μL from this dilution of fMLP and add it to a separate tube containing 297 μL RPMI (1:100).
- Add 250 μL of the second solution to 250 μL of basophil cell solution and incubate for 30 minutes at 37 °C.
RPMI: Unstimulated Control
- Add 250 μL RPMI to 250 μL basophil cell solution.
- Incubate for 30 minutes at 37 °C.
Anti-IgE
- Add 0.6 μL Anti-IgE to 300 μL RPMI
- Add 250 μL of this solution to 250 μL of basophil cell solution and incubate for 30 minutes at 37 °C.
Plate & Fix Basophils
Materials
- 30 ml PBS (Phosphate Buffered Saline)
- 30 ml 4% Paraformaldehyde in PBS (PFA-PBS); made from 5 ml stock PFA in 25 ml PBS
Procedure
- Once basophil cells are extracted from PBMC and stimulated, spin cells down at 300g for 7 minutes.
- Aspirate RPMI and add 50μL PBS to each tube.
- At 4 °C, apply ~50 μL basophil-PBS solution to center of Poly-L-Lysine-coated slides.
- Allow solution to sit for 15-20 minutes, undisturbed at 4 °C, so that cells can settle onto slide.
- Check with microscope to ensure cells adequately settled onto slide.
- Aspirate liquid from surface of slide, and wash each slide with 1mL PBS. To minimize cell loss allow PBS to run down the side of the well while washing.
- Aspirate and fix cells with 1 mL 4% PFA-PBS. Incubate at 4 °C in the dark for 15 minutes.
- Keep Cells on ice and in the dark until subsequent procedures are carried out.
Preparing Slides for Ab Tagging
Materials
- 15 mL PBS
- 100 mL 0.1% Triton X-100 PBS (TxPBS); made from 1 mL stock Triton X-100 in 100 mL PBS
- 15 mL Stain Buffer (1% bovine albumin in 2 mM EDTA in PBS)
Procedure
- Wash slides once with 1 mL PBS per well.
- Wash slides 3X with TxPBS. Each wash should incubate for 5 minutes between washes.
- Block non-specific binding by adding 1mL Staining Buffer. Incubate for 30 minutes at room temperature in the dark.
- Prepare Ab solution and apply as described in separate procedure.
mAb Staining
Materials
Procedure
- Overnight Staining with goat anti-CD203c (1:100 from 200 μg/mL stock); rabbit anti-CD107a (1:400 from 200 μg/mL) in staining buffer
- Next morning, wash 3 times for 5 minutes with TxPBS
- Stain with biotinylated donkey anti-goat (1:1000), incubate 1 hour.
- Wash 3 times for 5 minutes with TxPBS
- Prepare CD63 Ab
- For every 0.5 mL (1 SLIDE) add 4 μL mouse anti-CD63 (1:125) in 15 uL staining buffer, add 8 μL Zenon Alexa 594. Incubate 10 min at RT, add 8 μL Zenon blocking reagent. Incubate 10 min at RT.
- Stain with anti-CD63 cocktail, with 1:125 dilution of strepavidin Alexa 488 (for CD203c), and 1:400 donkey anti-rabbit 647 (for CD107a). Incubate for 1 hour.
- Wash 3 times for 5 minutes with TxPBS
- Fix with 4% PFA. Incubate for 15 minutes.
- Aspirate excess PFA, and mount slides with DAPI Vectashield mounting medium. Store at 4 °C in the dark until acquisition.
Plate Setup
A | B | C | |
1 | |||
2 | |||
3 | |||
4 |
Discussion
References
Contact
- Who has experience with this protocol?
- Email Alex Praslick through OpenWetWare