Dear Researchers, I would like to stain the intra cellular parasite of suspension cells especially theileria annulata. i have followed the procedure suggested by Dr Saleh Alkarim (Attached below) . But unfortunately, i could not able to stain the parasite and coudnt able to see the cytoplasm too. so kindly give some more protocol for the same.
Protocol which i followed :
1-Collect a cell suspension of 2 × 105 to 1 × 106 cells.
2-Pellet the cells by centrifugation and discard the supernatant.
3-Tap the tube to re-suspend the pellet in the residual liquid and add 1 mL of PBS at room temperature.
4-Transfer the full volume of re-suspended cells to 4 mL of absolute ethanol at –20°C by pipetting the cell suspension slowly into the ethanol while vortexing at top speed. Leave the cells in ethanol at –20°C for 5–15 minutes.
5-Pellet the cells by centrifugation and discard the ethanol.
6-Tap the tube to loosen the pellet and add 5 mL of PBS at room temperature. Allow the cells to rehydrate for 15 minutes.
Counterstaining protocol:
1-Dilute the DAPI stock solution to 3 µM in staining buffer (100 mM Tris, pH 7.4, 150 mM NaCl, 1 mM CaCl2, 0.5 mM MgCl2, 0.1% Nonidet P-40). A 1 mL volume will be required for each cell sample.
2-Centrifuge the cell suspension (from step 2.6) and discard the supernatant. Tap to loosen the pellet and add 1 mL of DAPI diluted in staining buffer.
3-Incubate for 15 minutes at room temperature.
4-Analyze by flow cytometry in the presence of the dye. If the cells are to be viewed by fluorescence microscopy, centrifuge the sample, remove the supernatant and resuspend cells in fresh buffer. Apply a drop of the suspension to a microscope slide, cover with a coverslip and view.
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