Shreffler:Confocal Protocols
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Confocal Protocols
. Other Project Page: BasophilConfocal
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)
Method
- 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 100μL 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
fMLP
- For first dilution (1:100), add 1µL fMLP to 99µL RPMI.
- Second dilution, take 3µL from this dilution of fMLP and add it to a separate tube containing 297µL RPMI (1:100).
- Add 250µL of the second solution to 250µL 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
Method
- 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)
Method
- 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.
CD63, CD203c, CD107a Staining
- 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.
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