Glycolysis capability (extracellular acidification) analysis.

Protocol for Glycolysis Capability Analysis (i.e., ECAR) for Histoplasma capsulatum

This document was prepared based on a request for a more detailed protocol for our method of glycolysis capability (extracellular acidification) analysis, as presented in Fig 3B of:

Shen Q, SC Ray, HM Evans, GS Deepe, Jr., and CA Rappleye. Metabolism of gluconeogenic substrates by an intracellular fungal pathogen circumvents nutritional limitations within macrophages. mBio. 2020; 11(2) 02712-19.

Special thanks to Kathryn Tormos and Nikhat Zaidi of Agilent Technologies for technical advice regarding the adaptation of the standard Seahorse protocol for use with Histoplasma capsulatum.

Outline:

1. General Notes
2. Specific protocol for Glycolysis Stress Test with H. capsulatum adapted to minimal media or intracellular environment
3. Protocol for Glycolysis Stress Test with H. capsulatum
4. Media Recipes

I. General Notes:

• “Hc” = Histoplasma capsulatum
• This protocol has been adapted from the Seahorse Glycolysis Stress Test assay (Agilent Technologies) for use with the yeast form of the fungal pathogen H. capsulatum, grow in suspension. This protocol represents a very rudimentary version of this assay (2-DG only) due to the constraints imposed by the difficulties in working with loosely adherent H. capsulatum.
• The originating protocol (optimized for adherent mammalian cells) is available on Agilent’s website. We highly recommend reading the Seahorse protocol to enhance understanding of the assay.
• Carryout all steps while taking appropriate biological safety precautions for H. capsulatum or other pathogens.
• We used an old Seahorse XF24 analyzer, with 24 well plates. This instrument is no longer supported by Agilent. We anticipate no difficulties in adapting this assay to use with a Seahorse XFe24 analyzer. Conversion to a 96-well plate format (XFe96) will require a pilot assay to titer the concentration of Hc.
• Assay media used in this protocol was consistent with requirements for Seahorse analysis, i.e., no strong buffers (like sodium bicarbonate), no phenol red (can act as a buffer at >3mg/L), and no FBS.
• H. capsulatum prefers a more acidic environment, thus this protocol uses a media pH of 7.0. Avoid using an even more acidic media, as the pH may drop below the recommended range (i.e., pH 6.4-7.4) over the course of the Seahorse analysis.
• Our primary troubleshooting difficulty was ensuring Hc adhered to the Seahorse assay plate. Dislodged Hc may not contribute to the ECAR reads, as it may be excluded from the microchamber created when the analyzer probe is lowered into the well. The motion of the probe in the well may be sufficient to fully dislodge loose Hc from the monolayer. After Seahorse analysis, inspect plates for significant disturbance of the Hc monolayer. It may be necessary to customize the assay program to limit the number of injections for the assay (i.e., here we limited the analysis to 1 injection, Port A = 2DG).
• Using Hc at the concentration reported here was sufficient for ECAR to reach the level of detection for the Seahorse analyzer. However, the oxygen consumption rate (OCR) at this concentration of Hc far exceeded the linear range recommended for Seahorse analysis of mitochondrial respiration. If you are interested in mitochondrial respiration, we recommend running a pilot assay to identify a more appropriate quantity of Hc perwell.
• Used a non-standard protocol:
  Measurement 1
  Mix: 00:03:00
  Wait: 00:02:00
  Measure: 00:03:00
  Cycles: 6
  Duration: 00:48:00
  
  Injection 1 Port: A = 2DG Mix: 00:03:00
  Wait: 00:02:00
  Measure 00:03:00
  Cycles: 3
  Duration: 00:24:00
   

II. Specific protocol for Seahorse Glycolysis Stress Test with H. capsulatum adapted to minimal media or intracellular environment.
 

Background:

Seahorse analysis requires glucose-supplemented media (i.e., 10 mM glucose) to measure glycolytic activity. A standard Seahorse assay requires a 1 h incubation of cells in Seahorse media (with glucose) in a non-CO2 incubator. However, our work was designed to compare the glycolytic capacities of H. capsulatum adapted to growth in minimal media or the intracellular environment. To avoid the effects of exposure to glucose during this 1 h period, we instead incubated Hc in a low volume of sterile water, then added concentrated Seahorse media (including glucose) immediately prior to running the analysis.
 

Preparation of Hc (Histoplasma capsulatum):

• Wash Hc slant in 1 mL Ham F-12 medium, then transfer to tube.
• Count Hc (prepare 1:100 dilution)
• Add 1.5 x 108 Hc to 50 mL Ham F-12 to a 125 mL glass culture flask with lid (3 x 106 per mL)
• Incubate Hc at 37°C in shaking water bath until Hc reaches exponential growth phase (approximately 48 h, monitor growth by OD595)

Adaptation of Hc to minimal media:

• Dilute Hc 1:5 into 3M minimal medium containing either 10 mM glucose or 10 mM casamino acids as the sole carbon source (see Media Recipes). Incubate at 37°C in shaking water bath for 24 h.

Growth of Hc in bone marrow-derived macrophages (BMDMs), i.e., “intramacrophage yeasts”:

• Briefly, bone marrow was flushed from hind limbs of 8-week-old wildtype mice (C57BL/6, Jackson Laboratory) and differentiated into macrophages (bone marrow-derived macrophages; BMDMs) by culturing in RPMI-1640 supplemented with 10% FBS, 0.1% gentamicin sulfate, 5 μM of 2-mercaptoethanol, and 10 ng/ml of mouse GM-CSF for 7 days at 37°C in 5% CO₂/95% air. Non-adherent cells were removed from tissue culture flasks by washing with PBS. BMDMs were scraped from the flask following an 8 min incubation in Trypsin.
• Plate BMDMs at 3 x 10⁶ cells per well in RPMI-1640 media with 10% FBS in 6-well standard tissue culture plates (i.e., a monolayer) and rest overnight at 37°C in 5% CO₂/95% air.
• Collect mid-log Hc from Ham F-12 medium, wash twice in HBSS (200 x g, 5 min), and resuspend in complete RPMI. “Slow spin” the Hc at 300 rpm x 5 min (approximately 11 x g) to loosely pellet large clusters of Hc, using the supernatant (predominately single yeasts or clusters of 2-3 yeast cells) for infection of BMDMs.
• Infect BMDMs with Hc at a multiplicity of infection (MOI) of 1 yeast per 2 BMDMs and incubate in RPMI-1640 media with 10% FBS at 37°C in 5% CO₂/95% air for 24 h.
• Lyse BMDMs in sterile water, collect intramacrophage yeast cells by centrifugation (400 × g for 5 min), and wash yeasts three times in water. Bring yeasts to a concentration of 4 x 10⁶ cells per 150 µl of water.

Day Before Assay:

• Prepare the calibration plate as described by Agilent:
  • Open the Agilent Seahorse XF24 Flux Assay Kit and remove the contents.
  • Place the sensor cartridge upside down next to the utility plate.
  • Fill each well of the utility plate with 1 mL of XF Calibrant.
  • Lower the sensor cartridge onto the utility plate, submerging the sensors in XF Calibrant.

• Verify the XF Calibrant level is high enough to keep the sensors submerged.
• Place in a non-CO₂ 37°C incubator overnight. To prevent evaporation of the XF Calibrant, the incubator should be humidified.

Seeding Hc on Seahorse cell culture plate and running the assay:

• Pre-treat Seahorse 24-well plate (Agilent) with 3 μg per well Poly-L-Lysine (3µg, 70-150 kDA, Sigma P4707) for five minutes at room temperature, rocking plate briefly to coat. Aspirate, then rinse wells in sterile water. Dry plate fully at room temperature before adding yeasts and media.
  • Stock = 0.01% w/v (aka 0.1 mg/mL) solution, Sigma P4707
  • Yeasts collected from glucose medium, casamino acid medium, or BMDMs were counted, then added onto a Seahorse culture plate pre-coated with poly-l-lysine at a quantity of 4 x 106 yeasts in 150 µl of water. Load 4 blank wells with 150 μl water, no Hc.
  • Centrifuge plate at 300 x g for 5 min to assist in adherence of Hc to the bottom of the well.
    Avoid any unnecessary disturbance of the plate, as the Hc may become dislodged.
  • Incubate plates in a non-CO₂ incubator at 37°C for 45 min to equilibrate (with Hc in 150 μl water).
    o See note on previous page – this was done in water to limit exposure of Hc to glucose in the Seahorse medium.
  • During the incubation:
    • Ensure program (see page 2) is set-up as desired in Seahorse analyzer.
    • Load Port A of the sensor cartridge with the glycolysis inhibitor 2-deoxy-D-glucose, 2-DG (Sigma Aldrich), for a final in-well concentration of 25 mM.
    • Begin calibration of the sensor cartridge as described in the official Seahorse protocol.
  • Immediately prior to analysis, gently and slowly add 350 μl of a 1.67x stock of glucose minimal medium, pH 7.0 (see recipe below) to all wells.
    • To avoid dislodging the Hc, add media slowly, with pipette tip against the side of the well.
    • Final concentration = 1x medium, 10 mM glucose, total volume 500 μl.
  • Load cell culture plate into analyzer and begin assay.
    • Tip: Confirm plate has successfully finished calibration steps (approx. 15-30 min) before leaving the lab. The read will stop if calibration fails (ex – pH out of range). If only a few wells fail, you can instruct the analyzer to proceed (later, exclude bad wells from the analysis).

Analysis:

• As this was a non-standard protocol, analysis will be more hands-on than typically required for a Seahorse assay. My work-around was as follows:
  • Exclude any bad wells in the Seahorse Wave software
  • Export data toExcel
  • Total ECAR = glycolytic ECAR + non-glycolytic acidification
    • Non-glycolytic acidification:
      • 2-DG inhibits glycolysis. Therefore, any remaining ECAR after the addition of 2-DG represents non-glycolytic acidification
      • Total ECAR is the ECAR reading before the addition of 2-DG (i.e., before glycolysis was inhibited). I prefer to use the reading immediately before the addition of 2-DG, as it is generally the most stable, but you may choose any earlier read.
      • To find the glycolytic ECAR, subtract the post-2-DG ECAR (i.e., non-glycolytic) from the initial (i.e., “total”) ECAR. With our instrument program, these reads occurred at approximately 44 min (pre-ECAR) and 70 min (post-ECAR)
         

III. Protocol for Glycolysis Stress Test with H. capsulatum (i.e., basic assay – standard media).

Note: This is not the assay reported in our publication. For this information, see section II. The publication used Hc adapted to nutrient-limited media and required additional steps to avoid extended exposure of the yeasts to rich medium prior to the Seahorse run.

Preparation of Hc (Histoplasma capsulatum):

• Wash Hc slant in 1 mL Ham F-12 medium, then transfer to tube.
• Count Hc (prepare 1:100 dilution)
• Add 1.5 x 108 Hc to 50 mL Ham F-12 to a 125 mL glass culture flask with lid (3 x 106 per mL)
• Incubate Hc at 37°C in shaking water bath until Hc reaches exponential growth phase (approximately 48 h, monitor growth by OD595)

Seeding Hc on Seahorse cell culture plate and running the assay:

• Pre-treat Seahorse 24-well plate (Agilent) with 3 μg per well Poly-L-Lysine (3µg, 70-150 kDA, Sigma P4707) for five minutes at room temperature, rocking plate briefly to coat. Aspirate, then rinse wells in sterile water. Dry plate fully at room temperature before adding yeasts and media.
  o Stock = 0.01% w/v (aka 0.1 mg/mL) solution, Sigma P4707
• Prepare Seahorse culture media. We suggest using the standard 1x Seahorse XF DMEM Medium (sold by Agilent) with pH adjusted to 7.0. Seahorse media is not well-buffered, so it is recommended that you confirm the pH of an aliquot (pre-warm to 37C) before eachrun.
• Yeasts collected from Ham F-12 were counted, then resuspended to 4 x 106 yeasts per 500 μl Seahorse media. Load 4 x 106 yeasts (500 μl) per well onto a 24-well Seahorse culture plate pre-coated with poly-l-lysine. Load 4 blank wells with 500 μl media, no Hc.
• Centrifuge plate at 300 x g for 5 min to assist in adherence of Hc to the bottom of the well.
  Avoid any unnecessary disturbance of the plate, as the Hc may become dislodged.
• Plates were incubated in a non-CO₂ incubator at 37°C for 45 min to equilibrate (with Hc in Seahorse media).
• During the incubation:
  o Ensure program (see page 2) is set-up as desired in Seahorse analyzer.
  o Load Port A of the sensor cartridge with the glycolysis inhibitor 2-deoxy-D-glucose, 2-DG (Sigma Aldrich), for a final in-well concentration of 25 mM.
  o Begin calibration of the sensor cartridge and calibration plate as described in the official Seahorse protocol.
• When calibration of sensor cartridge Load cell culture plate into analyzer and begin assay.
  o Tip: Confirm plate has successfully finished calibration steps (approx. 15-30 min) before leaving the lab. The read will stop if calibration fails (ex – pH out of range). If only a few wells fail, you can instruct the analyzer to proceed (later, exclude bad wells from the analysis).

Analysis: See Section II.
 

IV. Media Recipes

Minimal 3M media:

1X Yeast Nitrogen Base (w/o amino acids) 25 mM HEPES

16 mM Ammonium Chloride 100 uM Cystine

2 uM Thiamine

200 nM Biotin

20 uM Zinc Sulfate 10 uM Iron Sulfate

Notes:

1. Cystine stock: 70 mM cystine dissolved in 0.25M HCl
2. Iron Sulfate needs to be made fresh (stocks oxidize over time)
3. pH media to 6.0
4. filter sterilize
5. Add carbon source to media from sterile stock
   (or add powder to base media before filter sterilization): 10 mM Glucose → - or-  →10 mM Casamino Acids
    

Seahorse Assay Medium used in Section II:

Used during Seahorse run only. Hc was grown in minimal 3M media.

To replicate commercially available Seahorse DMEM media, use powdered DMEM without glucose, without L-glutamine, without phenol red, without pyruvate, without sodium bicarbonate (Sigma D5030).

Prepare a 1.67x stock, supplement with 16.7 mM glucose to 10 mM, adjust pH to 7.0, and filter sterilize. While incubating Hc for 1 h in the non-CO2 incubation, also incubate this media in a separate container that will allow dissipation of CO2, such as a standard tissue culture dish.

Q: Why 1.67x and 16.7 mM glucose?

A:    We add 350 μl of this media to Hc plated in 150 μl water per well, which combine for a total volume of 500 μl of 1x media, 10 mM glucose.

Seahorse Assay Medium used in Section III:

Commercially available 1x Seahorse XF DMEM medium (103575-100, Agilent) with pH adjusted to 7.0 (H. capsulatum prefers more acidic environments). Seahorse media is not well-buffered, so it is recommended that you confirm the pH of an aliquot (pre-warm to 37°C) before each run.

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