could be because (i) HIF1 is already blocked and Co2+ has no effect or (ii) it is already induced in your system and Co2+ does not increase it further.

To test this, I would try other cell line(s) .

As for hypoxic conditions, you may put your cells to a flask (regular cap), then pump some N2 real quick, close the cap and incubate the flask for a couple hours - it is normally enough to induce HIF but not yet lethal for the cells.

To establish hypoxia conditions in a low cost chamber, you can follow this research paper.

Article Engineering oxygen nanobubbles for the effective reversal of hypoxia

We used a very simple protocol to create hypoxia conditions as you can read in the paper. CoCl2 induces chemical changes and not real lack of oxygen, therefore, its a good indicator of HIF1a, but its not real lack of oxygen and cell breathing is normal.

Also, HIF-1a will express under normal conditions also. Especially if your cell confluence is high, (80% or more confluence), you are likely to have high values of HIF`1a.

You can refer to our another paper for HIF1a expression during in vivo conditions.

Article Effective delivery of mycophenolic acid by oxygen nanobubble...

May be the cells you are using overexpress HIF 1 from the beginning. Sort of Von Hippel Lindau disease.

Try to use another cell line and see what happens. Another possibility is to check your cells for VHL protein.

Thank you all for your valuable response.