6A(v) |
No visible cells in the interface |
Low amount of starting material |
Collect the interface together with Biocoll solution |
6A(ix) |
Low yield of isolated cells |
Low amount of starting material |
Increase the number of animals; use older or larger animals |
Insufficient dissociation of kidney marrow |
Dissociate the marrow more extensively, and split kidney marrow from multiple animals into several tubes |
6B(vi) |
Low number of sorted cells |
Rare population of gated cells |
Change the gating strategy |
Cells do not enter the sorting tube |
Recalibrate the FACS instrument |
Cells stick to the wall of the sorting tube, dying during the sorting process |
Coat the wall of collection tubes with medium, and re-coat it during the sort if needed |
Cells die during the sorting process |
Make sure to keep the cells chilled during the sorting process |
12 |
No or low colony growth |
Low number of seeded cells |
Increase the number of seeded cells |
Ineffective cytokine stimulation |
Increase the effective concentration of cytokine tested or test cooperation between several cytokines |
Unhealthy or inbred fish |
Test different fish |
Methylcellulose too dense |
Prepare fresh 2% (wt/vol) methylcellulose stock and mix new complete methylcellulose |
Colonies are present even in a negative control |
Colony density is too high |
Plate fewer cells; when over-plated, cells often generate microenvironments that help them survive and proliferate even without the presence of exogenous supporting factors |
Yeast or bacterial contamination producing microbial colonies |
Follow sterile techniques, especially during kidney dissection; use sterile forceps when removing kidney marrow; test for the presence of microbial organisms in a culture medium, especially in carp serum |
Large chunks present in methylcellulose |
Methylcellulose stock contains insoluble pieces of methocel |
Swirl methylcellulose vigorously after bringing it to a boil during its preparation. Allow the methylcellulose stock to sediment for 1–2 d before using it for mixing the complete methylcellulose. Note: it is common for methylcellulose to contain a small amount of insoluble clumps |
Cells are concentrated on a single area within the well |
Inefficient mixing of cell– methylcellulose mixture |
Mix the cell–methylcellulose mixture more thoroughly |
13 |
Inability to isolate single colonies |
Colony density is too high |
Plate fewer cells |